Wassila Riah

Karine Laval

Emilie Laroche-Ajzenberg

Christian Mougin

Xavier Latour

Isabelle Trinsoutrot-Gattin

Effects of pesticides on soil enzymes: a review

Keywords: Pesticides, Soil, Enzyme activity, Microbial diversity, Microcosm studies

The use of pesticides in agriculture has highly increased during the last 40 years to increase crop yields. However, today most pesticides are polluting water, soil, atmosphere and food.

Pesticides are also impact soil enzymes, which are essential catalysts ruling the quality of soil life. In particular, the activity of soil enzymes control nutrient cycles, and, in turn, fertilization.

Here, we review the effects of pesticides on the activity of soil enzymes in terrestrial ecosystems. Enzymes include dehydrogenase, fluorescein diacetate hydrolase, acid phosphatase, alkaline phosphatase, phosphatase, -glucosidase, cellulase, urease and arylsulfatase. Those enzymes are involved in the cycles of carbon, nitrogen, sulfur and phosphorus.

The main points of our analysis are (1) the common inhibition of dehydrogenase in 61 % of studies, stimulation of cellulase in 56 % of studies and no response of aryl-sulfatase in 67 % of studies. (2) Fungicides have mainly negative effects on enzymatic activities. (3) Insecticides can be classified into two groups, the first group represented by endosulfan having an overall positive impact while the second group having a negative effect. (4) Herbicides can be classified into two groups, one group with few positive effect and another group with negative effect.

Introduction

Pesticides are widely used in crop production and are known to induce major environmental problems in Europe. With an increased pesticide use, questions are rising on potential effects regarding public health and environment. Pesticides pollute air, soil, water resources and contaminate the food chain. The European Commission has taken into consideration this problem by publishing the Directive 2009/128/EC, which establishes a framework to achieve a sustainable use of pesticides. This directive aims at reducing the risks and impacts of pesticide use on human health and environment by promoting the use of integrated pest management and alternative approaches or techniques such as non-chemical alternatives to pesticides.

Although the Directive 91/414/EEC of 15 July 1991 on the authorization of plant protection products has already resulted in the elimination of more than 75 % of active substances from the market according to their ineffective-ness or toxicity [START_REF] Karabelas | Impact of European legislation on marketed pesticides: a view from the standpoint of health impact assessment studies[END_REF]), a large variety of active molecules are still used. Over 150 different pesticides are currently authorized in member states of the European Union. In agriculture, these compounds are applied to improve crop yield and quality. Globally, about 140.000 tons of pesticides are applied annually in the Europe (Eurostat 2007). The European farmers devote a significant portion of their budget to buy these agrochemicals. Usually, several pesticides are required during a cropping season, thus agricultural soils often contain a mix of different pesticides at different concentrations.

Pesticides include diverse groups of inorganic and organic chemicals. They are divided into groups according to their primary target and include herbicides, insecticides, nematicides, fungicides and soil fumigants [START_REF] Gevao | Bound pesticide residues in soils: a review[END_REF]. Pesticides can be classified in a number of ways: by target pest, their mode of action or chemical family. The main chemical groups are organochlorine, organophosphate, carbamate, pyrethroids, triazine and sulfonylurea [START_REF] Afify | Multiresidue method of analysis for determination of 150 pesticides in grapes using quick and easy method (QuEChERS) and LC-MS/MS determination[END_REF]. There are a large variety of pesticides available on the market, and chemical families are more diversified than proposed by these authors (Table 1). Some chemical families of pesticides can be efficient as fungicide, herbicide and insecticide, which make it difficult to classify them.

The interaction between soil components and pesticides influences the biochemical processes driven by microorganisms. Telluric fungi [START_REF] Hernandez-Rodriguez | Degradation of endosulfan during substrate preparation and cultivation of Pleurotus pulmonarius[END_REF][START_REF] Ronhede | Mineralization of hydroxylated isoproturon metabolites produced by fungi[END_REF]) and bacteria [START_REF] Dong | Crystal structure of methyl parathion hydrolase from Pseudomonas sp WBC-3[END_REF][START_REF] Qiu | Isolation and characterization of a bacterial strain of the genus Ochrobactrum with methyl parathion mineralizing activity[END_REF]) are able to degrade or mineralize pesticides via enzymatic reactions. Pesticides' effects on soil microorganisms can be determined by the study of functional parameters such as carbon and nitrogen mineralization that are governed by enzymatic activities. Those activities play an important role because all biochemical transformations in soil depend on or are related to the presence of enzymes. They are indicators of biological equilibrium [START_REF] Frankenberger | Factors affecting L-asparaginase activity in soils[END_REF], fertility (Schuster and Schroder 1990a b;[START_REF] Antonious | Impact of soil management and two botanical insecticides on urease and invertase activity[END_REF] and changes in the biological status due to soil pollution [START_REF] Nannipieri | Use of enzymes to detoxify pesticide-contaminated soils and waters[END_REF][START_REF] Kucharski | Response of soil microorganisms and buckwheat plants to cytokinines[END_REF][START_REF] Trasar-Cepeda | Limitations of soil enzymes as indicators of soil pollution[END_REF][START_REF] Chu | Effects of lanthanum on dehydrogenase activity and carbon dioxide evolution in a Haplic Acrisol[END_REF][START_REF] Bending | Microbial and biochemical soil quality indicators and their potential for differentiating areas under contrasting agricultural management regimes[END_REF]). Finally, the measurement of specific enzymatic activities may contribute to understand the metabolic processes involved in the biogeochemical cycles of nutrients. Pesticides reaching the soil may disturb local metabolism or enzymatic activities [START_REF] Engelen | Monitoring impact of a pesticide treatment on bacterial soil communities by metabolic and genetic fingerprinting in addition to conventional testing procedures[END_REF][START_REF] Liu | Distribution of soil enzyme activities and microbial biomass along a latitudinal gradient in farmlands of songliao plain, Northeast China[END_REF][START_REF] Hussain | Impact of pesticides on soil microbial diversity, enzymes, and biochemical reactions[END_REF]. Negative impacts of pesticides on soil enzymes such as hydrolases, oxidoreductases and dehydrogenase activities have been widely reported in the literature [START_REF] Perucci | Effects of the herbicide imazethapyr on soil microbial biomass and various soil enzyme-activities[END_REF][START_REF] Ismail | Effects of metsulfuron-methyl on amylase, urease, and protease activities in two soils[END_REF][START_REF] Malkomes | Effects of steaming and pesticides on soil microorganisms under laboratory conditions. II. Effects of partial sterilization and its combination with pesticides[END_REF]Monkiedje and Spiteller 2002;Monkiedje et al. 2002;[START_REF] Menon | Effects of chlorpyrifos and quinalphos on dehydrogenase activities and reduction of Fe3? in the soils of two semi-arid fields of tropical India[END_REF][START_REF] Caceres | Effect of insecticide fenamiphos on soil microbial activities in Australian and Ecuadorean soils[END_REF]. There is also evidence that soil enzymes may pro-vide valuable general information on transformation of pesticides in soils [START_REF] Gianfreda | Effect of soils on the behavior of immobilized enzymes[END_REF][START_REF] Kalam | Pesticide effects on microbial population and soil enzyme activities during vermicomposting of agricultural waste[END_REF][START_REF] Gil-Sotres | Different approaches to evaluating soil quality using biochemical properties[END_REF][START_REF] Hussain | Impact of pesticides on soil microbial diversity, enzymes, and biochemical reactions[END_REF].

Once a pesticide is released into the environment, understanding its behavior becomes of major scientific interest. Its evolution is strongly linked to soil's microbiological composition and especially to soil enzymatic activities. The diversity of pedoclimatic contexts, pesticides' nature and experimental protocols (applied doses) in scientific literature make data analysis extremely complex [START_REF] Gevao | Bound pesticide residues in soils: a review[END_REF]. Nevertheless, the literature in this area is abundant and the authors often provide findings that suggest that enzymatic activities could be indicators of soil use and management because of their relationship to soil biology [START_REF] Yao | Influence of acetamiprid on soil enzymatic activities and respiration[END_REF]. Soil enzymatic activities are assumed to be early indicators of soil degradation compared to chemical or physical parameters [START_REF] Dick | Soil enzyme-activities after 1500 years of terrace agriculture in the Colca valley, Peru[END_REF]). Among soil enzymatic activities, hydrolases are the most commonly measured activities in soils and therefore proposed by many authors as potential indicators of soil state [START_REF] Tabatabai | Soil enzymes[END_REF][START_REF] Deng | Effect of tillage and residue management on enzyme activities in soils: II. Glycosidases[END_REF][START_REF] Dick | Soil enzyme activities and biodiversity measurements as integrative microbiological indicators[END_REF][START_REF] Floch | Indicators of pesticide contamination: soil enzyme compared to functional diversity of bacterial communities via Biolog Ecoplates[END_REF].

The main aim of this work is to summarize and analyze the rich data accumulated in the literature during the last years, concerning pesticide effects on soil enzymatic activities: dehydrogenase and a large range of hydrolases. This review attempts to find out or identify common determinants explaining variation patterns of soil enzymatic activities in relationship with application of different types of pesticides. The patterns of soil enzymatic responses could be then used to reflect experimental designs (most relevant enzymatic activities to be monitored, pesticide doses to be applied…) aiming at understanding the impact of pesticides on soil microbes. More generally, they could provide valuable support for decision-making (farmers, advisers, public authorities) concerning the choice of pesticides, which are less harmful for soil microorganisms.

Here, we review the effects of pesticides on enzymatic activities and we discuss trends of response for each soil enzymes. We also analyze the possible relationships between the responses of soil enzymatic and mechanisms action pesticides.

Effect of pesticides on soil enzymatic activities

The literature concerning the effect of pesticides on soil enzymatic activities is abundant and sometimes discordant. Some studies were realized in field while others were performed in natural microcosms. In the latter case, soil preparation and incubation times were very variable in function of authors. Thereby, in order to achieve a comparative analysis, we have chosen to examine only the data that refer to microcosm experiments (Fig. 1). These con-trolled experiments have indeed the advantage to provide more robust results; the small ecosystem size enables high replication in experiments [START_REF] Srivastava | Are natural microcosms useful model systems for ecology?[END_REF]. Soil microcosms allow also observing the effects of only one pesticide on soil enzymatic activities and not the synergic or antagonist effects of molecules, as it is often the case in field studies where it is difficult to dispense of cumulative effects of pesticides resulting of agricultural practices. Moreover, only the results observed between 28 and 50 days of incubation were reported here, in order to avoid considering transient effects of pesticides on enzymatic activities.

The synthesis of the review is reported in Table 2. It highlights that the enzymes studied are either indicators of overall microbiological activity, or specific hydrolases of carbon, nitrogen, phosphorus and sulfur biochemical cycles. In the different studies, the applied doses of pesticides were given in reference to the field rate (FR, i.e., the approved dose). It appears that many studies used 10, 100 up to 1,000 times the recommended field dose in order to simulate repeated application or long-term use of pesticides, even if this method is questionable.

Global metabolic activities

Dehydrogenase activity

Dehydrogenase occurs in all living microbial cells, and it is linked with microbial respiratory processes [START_REF] Bolton | Soil microbial biomass and selected soil enzyme-activities: effect of fertilization and cropping practices[END_REF]. This intracellular enzyme is an indicator of overall microbial activity of soils. The impact of pesticides on dehydrogenase activity has been widely reported in the literature. Pesticides generally appear to have an adverse effect on dehydrogenase activity (Table 2). The majority of insecticides are either neutral toward this activity [START_REF] Caceres | Effect of insecticide fenamiphos on soil microbial activities in Australian and Ecuadorean soils[END_REF] or they inhibit it [START_REF] Beulke | Effects of the herbicides metazachlor and dinoterb on the soil microflora and the degradation and sorption of metazachlor under different environmental conditions[END_REF][START_REF] Kalam | Pesticide effects on microbial population and soil enzyme activities during vermicomposting of agricultural waste[END_REF][START_REF] Yao | Influence of acetamiprid on soil enzymatic activities and respiration[END_REF][START_REF] Jastrzebska | The effect of chlorpyrifos and teflubenzuron on the enzymatic activity of soil[END_REF]. Only endosulfan seems to stimulate dehydrogenase activity when it goes 100 to 200 times the standard rate of application [START_REF] Kalyani | Influence of endosulfan on microbial biomass and soil enzymatic activities of a tropical Alfisol[END_REF][START_REF] Defo | Microcosm study of the long term effect of endosulfan on enzyme and microbial activities on two agricultural soils of Yaounde-Cameroon[END_REF]). But con-tradictory results show no effect of endosulfan at 200 times the regular dose in soil with pH 4.8. Likewise, herbicides, except butachlor [START_REF] Min | Effects of butachlor on microbial enzyme activities in paddy soil[END_REF][START_REF] Xia | Influence of butachlor on soil enzymes and Microbial growth[END_REF], have a repressive effect on dehydrogenase activity, whatever conditions of application, including dose and soil pH [START_REF] Beulke | Effects of the herbicides metazachlor and dinoterb on the soil microflora and the degradation and sorption of metazachlor under different environmental conditions[END_REF][START_REF] Bennicelli | Influence of pesticide (glyphosate) on dehydrogenase activity, pH, Eh and gases production in soil (laboratory conditions)[END_REF][START_REF] Sebiomo | Effect of four herbicides on microbial population, soil organic matter and dehydrogenase activity[END_REF]. In contrast, it was not possible to identify a single type of response of this activity to fungicides: the enzyme was alternately stimulated and inhibited. Authors suggested that the absence of effect on dehydrogenase activity was probably due to the time of incubation, which was insufficient to induce any effect. Nevertheless, the effect of a pesticide on soil microorganisms is governed not only by the chemical and physical properties of the pesticide itself, but also by the soil type, soil properties, and prevailing environmental conditions [START_REF] Dick | Soil acid and alkaline phosphatase activity as pH adjustment indicators[END_REF]. The dehydrogenase was severely inhibited at higher doses of fungicides (Monkiedje et al. 2002;[START_REF] Bello | Evaluation of various tests for the diagnosis of soil contamination by 2,4,5-trichlorophenol (2,4,5-TCP)[END_REF].

Generally, whatever the dose considered, fungicides, herbicides and insecticides show inhibitory effects or no effects on the dehydrogenase activity, except endosulfan and mancozeb.

Fluorescein diacetate hydrolase

The fluorescein di-acetate hydrolase activity has the potential to broadly represent soil enzymatic activities and accumulated biological effects. Fluorescein di-acetate is a substrate hydrolysed by a number of different enzymes, such as protease, lipase and esterase and its hydrolysis was observed among a wide array of primary decomposers, bacteria and fungi [START_REF] Janvier | Soil health through soil disease suppression: which strategy from descriptors to indicators?[END_REF]). In comparison with dehydrogenase, only few studies have examined the response of fluorescein diacetate hydrolase activity to the presence of pesticides in soil. There is no clear answer of fluorescein diacetate hydrolase to pesticides' input, but it seems to be more influenced by insecticides [START_REF] Das | Effect of novaluron on microbial biomass, respiration, and fluorescein diacetate-hydrolyzing activity in tropical soils[END_REF][START_REF] Bishnu | Ethion degradation and its correlation with microbial and biochemical parameters of tea soils[END_REF]) than herbicides [START_REF] Perucci | Effects of organic amendment and herbicide treatment on soil microbial biomass[END_REF][START_REF] Zabaloy | An integrated approach, to evaluate the impacts of the herbicides glyphosate, 2,4-D and metsulfuron-methyl on soil microbial communities in the Pampas region, Argentina[END_REF]. Fluorescein diacetate hydrolase activity was stimulated by the supply of pesticides of imidazolines (Imazethapyr) and organochlorines (endosulfan) families [START_REF] Perucci | Effects of organic amendment and herbicide treatment on soil microbial biomass[END_REF][START_REF] Kalyani | Influence of endosulfan on microbial biomass and soil enzymatic activities of a tropical Alfisol[END_REF], which have been less studied than other families of pesticides. The application of organophosphate (chlorpyrifos and ethion) at different doses had the same effect on this enzymatic activity [START_REF] Dutta | Effect of chlorpyrifos on microbial biomass and activities in tropical clay loam soil[END_REF][START_REF] Bishnu | Ethion degradation and its correlation with microbial and biochemical parameters of tea soils[END_REF].

Unfortunately, the number of experimentations carried out was not sufficient to draw any conclusion. It would be interesting to examine whether the response of fluorescein diacetate hydrolase is the same with more represented organophosphate pesticides. Slight and transitory increases in fluorescein diacetate hydrolase activity were observed at the highest applied pesticide rates (tenfold field rate).

Moreover, the author proposed a new synthetic index, the specific hydrolytic activity, to assess microbial activity in reply to xenobiotic treatments, and he considered fluores-cein diacetate hydrolase as a suitable tool for measuring the early detrimental effect of pesticides on soil microbial biomass, as it is a sensitive and non specific test, which is able to depict the hydrolytic activity of soil microbes. No study on the effects of fungicides on fluorescein diacetate hydrolase activity has been identified in this review. Fluorescein diacetate hydrolase activity in soil is poorly influenced by herbicides or insecticides applications, except endosulfan applications, which seems to stimulate this activity.

Carbon cycle enzymatic activities

Cellulase and -glucosidase -Glucosidase and cellulase are very important enzymes involved in the transformation/decomposition of organic matter in soil. Their final product is glucose, an important carbon energy source for soil microorganisms [START_REF] Deng | Cellulase activity of soils[END_REF].

Very few references are available since the year 2000 concerning the effect of fungicides on cellulase activity. Indeed, only two fungicides have been tested and seem to have no pronounced negative effects on this activity [START_REF] Niemi | Microbial toxicity and impacts on soil enzyme activities of pesticides used in potato cultivation[END_REF]. Likewise, the herbicides from different chemical families (urea, triazine and nitrile) seem to have no effect on cellulase activity even with 10 times the dose of application [START_REF] Omar | Microbial populations and enzyme activities in soil treated with pesticides[END_REF][START_REF] Niemi | Microbial toxicity and impacts on soil enzyme activities of pesticides used in potato cultivation[END_REF]). The insecticides are more represented, particularly molecules, which belong to the family of organophosphate (monocrotophos, quinalphos, profenofos and selectron). They have globally a stimulating effect on this enzymatic activity [START_REF] Omar | Microbial populations and enzyme activities in soil treated with pesticides[END_REF][START_REF] Gundi | Activities of cellulase and amylase in soils as influenced by insecticide interactions[END_REF][START_REF] Niemi | Microbial toxicity and impacts on soil enzyme activities of pesticides used in potato cultivation[END_REF]). These molecules may destroy soil insects and make substrates available to stimulate cellulase activity. Moreover, [START_REF] Gundi | Activities of cellulase and amylase in soils as influenced by insecticide interactions[END_REF] showed that there was a link between enzymatic activity of cellulase in soils and cellulolytic fungi in the presence of insecticides. Indeed Populations of cellulolytic bacteria in both the black vertisol and red alfinsol soils were enhanced with increasing concentration of monocrotophos, quinalphos and profenofos. The cellulase activity is either inhibited or insensitive to the fungicides and herbicides tested, while the various insecticides tested, except Selectron, have a stimulatory effect on the enzyme's activity.

According to the data presented in Table 2, the activity of -glucosidase shows two patterns of variation in the presence of pesticides: it is either inhibited or stays unchanged. The fungicides from amides' family (mefenoxam et metalaxyl) affect the activity of the glucosidase (Monkiedje et al. 2002). However, the combined supply of a fumigant (methyl bromide) and a fungicide (chloropricin) has no effect on this activity. It was observed that herbicides have no effect on -glucosidase activity, even when using molecules, which belong to different herbicides' families (linuron and metribuzin) [START_REF] Niemi | Microbial toxicity and impacts on soil enzyme activities of pesticides used in potato cultivation[END_REF]). On the contrary, it appears that glyphosate and diflufenican applied to a same soil inhibit the -glucosidase activity [START_REF] Tejada | Evolution of soil biological properties after addition of glyphosate, diflufenican and glyphosate plus diflufenican herbicides[END_REF]). -glucosidase activity do not show a clear response to insecticides, except endosulfan, which seems to stimulate the activity of -glucosidase at 200 times the usual dose [START_REF] Defo | Microcosm study of the long term effect of endosulfan on enzyme and microbial activities on two agricultural soils of Yaounde-Cameroon[END_REF]. It seems that the -glucosidase activity is either inhibited or insensitive to the application of pesticides, whether fungicides, insecticides or herbicides, except for the results observed by [START_REF] Defo | Microcosm study of the long term effect of endosulfan on enzyme and microbial activities on two agricultural soils of Yaounde-Cameroon[END_REF] with endosulfan on acid soil. This observation may be related to the strong functional redundancy of -glucosidase activity, since a large number of microbial species, whether fungi or bacteria, is able to express this enzymatic activity.

Phosphorus cycle enzymatic activities

Acid phosphatase, alkaline phosphatase and phosphatases Phosphatases include five major groups of enzymes: The phosphomonoesterases, the phosphodiesterases, the phosphotriesterases, the pyrophosphatases and the phosphoamidases. Among these enzymes, phosphomonoesterases are the most abundant in soils, probably due to the low substrate specificity of this group of enzymes [START_REF] De Cesare | Use of sonification for measuring acid phosphatase activity in soil[END_REF].

Phosphomonoesterases include acid and alkaline phosphatase, which can be distinguished according to the optimum pH for their activity. In literature, some authors use the term phosphatase without distinguishing between acid and alkaline phosphatase. Phosphatases are a broad group of enzymes that are capable of catalysing hydrolysis of esters and anhydrides of phosphoric acid [START_REF] Schmidt | The distribution of alkaline phosphatase in the regenerating forelimb of the adult newt, Diemictylus viridescens (Triturus v.)[END_REF]Schmidt et al. 1962). In soil ecosystems, these enzymes are believed to play a critical role in Phosphorus cycle [START_REF] Schneider | Phosphatase activity, microbial phosphorus, and fine root growth in forest soils in the Sierra de Gata, western central Spain[END_REF].

Experi-ments show that they are correlated to phosphorus stress and plant growth. Apart from being good indicators of soil fertility, phosphatase enzymes play a key role in the soil system [START_REF] Eivazi | Phosphatases in soils[END_REF][START_REF] Dick | Factors affecting hydrolysis of polyphosphates in soils[END_REF][START_REF] Dick | Soil acid and alkaline phosphatase activity as pH adjustment indicators[END_REF]. The effect of pesticides on phosphatases, in particular on acid and alkaline phosphatase activities (when the distinction is made by the authors), is given in Table 2. Several researchers have shown either unchanged or decreasing phosphatase activity following various pesticide applications [START_REF] Kalam | Pesticide effects on microbial population and soil enzyme activities during vermicomposting of agricultural waste[END_REF][START_REF] Yan | Dissipation of carbendazim and chloramphenicol alone and in combination and their effects on soil fungal: bacterial ratios and soil enzyme activities[END_REF]. Acid and alkaline phosphatases are mostly found in microorganisms and animals [START_REF] Tabatabai | Alkaline-phosphatase activity and michaelis constant in the presence of different clay-minerals -comment[END_REF]). It appears that the supply of fungicides inhibits alkaline phosphatase activity [START_REF] Rasool | Effect of the fungicide Mancozeb at different application rates on enzyme activities in a silt loam soil of the Kashmir Himalaya, India[END_REF][START_REF] Sharma | Effect of pesticides on biochemical properties of tomato (Solanum lycopersicon) cropped soil[END_REF], whereas it stimulates the activity of acid phosphatase. This observation is more pronounced in the study conducted by Monkiedje et al. (2002), which showed that the application of mefenoxam and metalaxyl fungicides in soil at pH 7.2 inhibited alkaline phosphatase activity and stimulated acid phosphatase activity. Regarding the phosphatases, only the fungicide metalaxyl seems to stimulate this group of enzymes [START_REF] Sukul | Enzymatic activities and microbial biomass in soil as influenced by metalaxyl residues[END_REF]. Other fungicides either had no effect or inhibited their activity [START_REF] Bello | Evaluation of various tests for the diagnosis of soil contamination by 2,4,5-trichlorophenol (2,4,5-TCP)[END_REF][START_REF] Tejada | Effects of Prochloraz fungicide on soil enzymatic activities and bacterial communities[END_REF][START_REF] Yan | Dissipation of carbendazim and chloramphenicol alone and in combination and their effects on soil fungal: bacterial ratios and soil enzyme activities[END_REF]. Regarding the herbicides, most of the time the response of acid and alkaline phosphatase activity is similar, and the two activities are either stimulated (imazethapyr) or remain unchanged (aurora 40WG and rimsulfuron) [START_REF] Perucci | Effects of organic amendment and herbicide treatment on soil microbial biomass[END_REF][START_REF] Omar | Microbial populations and enzyme activities in soil treated with pesticides[END_REF][START_REF] Bacmaga | Enzymatic activity in soil contaminated with the Aurora 40 Wg herbicide[END_REF]).

Butachlor appears to stimulate the activity of alkaline phosphatase [START_REF] Xia | Influence of butachlor on soil enzymes and Microbial growth[END_REF]. Herbicides severely inhibit phosphatase activities even when they are applied in very different conditions in regard to the pesticides dose and soil physicochemical properties [START_REF] Min | Effects of butachlor on microbial populations and enzyme activities in paddy soil[END_REF][START_REF] Tejada | Evolution of soil biological properties after addition of glyphosate, diflufenican and glyphosate plus diflufenican herbicides[END_REF]. The enzymatic activities of acid and alkaline phosphatase respond differently to insecticides. Indeed, the same insecticide may inhibit acid phosphatase and stimulate alkaline phosphatase activity, and vice versa [START_REF] Omar | Microbial populations and enzyme activities in soil treated with pesticides[END_REF][START_REF] Cycon | Responses of indigenous microorganisms to a fungicidal mixture of mancozeb and dimethomorph added to sandy soils[END_REF][START_REF] Defo | Microcosm study of the long term effect of endosulfan on enzyme and microbial activities on two agricultural soils of Yaounde-Cameroon[END_REF][START_REF] Jastrzebska | The effect of chlorpyrifos and teflubenzuron on the enzymatic activity of soil[END_REF]. The difference in behavior of both acid and alkaline phospha-tases toward pesticides can be attributed to the structure of soil microbial communities and their sensitivity to pesti-cides applications [START_REF] Klose | Microbial community composition and enzyme activities in a sandy loam soil after fumigation with methyl bromide or alternative biocides[END_REF]. Insecticides had inhibitory effects on phosphatases [START_REF] Madhuri | Influence of selected insecticides on phosphatase activity in groundnut (Arachis hypogeae L.) soils[END_REF][START_REF] Yao | Influence of acetamiprid on soil enzymatic activities and respiration[END_REF]). However, the cadusaphos (organophosphate insecticide) applied at 10 times the recommended dose seemed to have no effect on phosphatase activities [START_REF] Vavoulidou | Response of soil quality indicators to the pesticide cadusaphos[END_REF]. Overall, pesticides appear to have an inhibitory effect on the enzymatic activities involved in the phosphorus cycle.

Nitrogen cycle enzymatic activity

Urease

Urease is an enzyme that catalyses the hydrolysis of urea into carbon dioxide and ammonia and is a key component in the nitrogen cycle in soils. Due to this role, urease activities in soils have received a lot of attention since it was first reported by [START_REF] Rotini | La trasformazione enzimatica dell'urea nel terreno[END_REF], a process considered vital in the regulation of nitrogen supply to plants after urea fertilization [START_REF] Makoi | Selected soil enzymes: examples of their potential roles in the ecosystem[END_REF]. Soil urease originates mainly from plants (Polacco 1977a, b) and microorganisms. It is found both as intra-and extra-cellular enzyme [START_REF] Blakeley | Jack-Bean-Urease: The 1 st nickel enzyme[END_REF][START_REF] Burns | Blockage and recovery of nitrification in soils exposed to acetylene[END_REF][START_REF] Mobley | Microbial ureases: significance, regulation, and molecular characterization[END_REF]. Most of the referenced studies reported that herbicides and fungicides appear to have no effect [START_REF] Cycon | Responses of indigenous microorganisms to a fungicidal mixture of mancozeb and dimethomorph added to sandy soils[END_REF][START_REF] Romero | Enzyme activities and diuron persistence in soil amended with vermicompost derived from spent grape marc and treated with urea[END_REF][START_REF] Tejada | Effects of Prochloraz fungicide on soil enzymatic activities and bacterial communities[END_REF][START_REF] Yan | Dissipation of carbendazim and chloramphenicol alone and in combination and their effects on soil fungal: bacterial ratios and soil enzyme activities[END_REF][START_REF] Bacmaga | Enzymatic activity in soil contaminated with the Aurora 40 Wg herbicide[END_REF] or reduced effect on urease activity [START_REF] Sukul | Enzymatic activities and microbial biomass in soil as influenced by metalaxyl residues[END_REF][START_REF] Caceres | Effect of insecticide fenamiphos on soil microbial activities in Australian and Ecuadorean soils[END_REF][START_REF] Tejada | Evolution of soil biological properties after addition of glyphosate, diflufenican and glyphosate plus diflufenican herbicides[END_REF]. Decreased urease activity in soil due to the application of pesticides reduces urea hydrolysis, which is generally beneficial, because it helps to maintain nitrogen availability to plants [START_REF] Antonious | Impact of soil management and two botanical insecticides on urease and invertase activity[END_REF]. On the contrary, the fungicides carbendazim and validamycin enhanced urease activity, respectively, up to 70 % and to 13-21 % [START_REF] Qian | Effects of validamycin on some enzymatic activities in soil[END_REF][START_REF] Yan | Dissipation of carbendazim and chloramphenicol alone and in combination and their effects on soil fungal: bacterial ratios and soil enzyme activities[END_REF]. The urease activity appears to be either unaffected or inhibited by the addition of pesticides except carbendazim and validamycin, which tend to stimulate this enzyme activity. Thus, it is difficult to identify a clear response of this enzymatic activity to pesticides because this enzyme has received little attention during the last 10 years.

Sulfur cycle enzymatic activity

Arylsulfatase

Arylsulfatases are typically widespread in soils (Tabataba and Bremner 1970b, c;[START_REF] Gupta | Distribution of microbial biomass and its activity in different soil aggregate size classes as affected by cultivation[END_REF][START_REF] Ganeshamurthy | Arylsulfatase and the biochemical mineralization of soil organic sulfur[END_REF][START_REF] Ganeshamurthy | Residual management of sulfur applied to soybean or wheat in a soybean-wheat system on Vertisols[END_REF]. They are responsible for the hydrolysis of sulfate esters in the soil [START_REF] Kertesz | The role of soil microbes in plant sulphur nutrition[END_REF] and are secreted by bacteria into the external environment as a response to sulfur limitation [START_REF] Mcgill | Comparative aspects of cycling of organic C, N, S and P through soil organic-matter[END_REF].The effects of pesticides on arylsulfatase activity in soil are poorly documented from the year 2000. Generally, the pesticides do not seem to affect the activity of this enzyme [START_REF] Niemi | Microbial toxicity and impacts on soil enzyme activities of pesticides used in potato cultivation[END_REF][START_REF] Tejada | Evolution of soil biological properties after addition of glyphosate, diflufenican and glyphosate plus diflufenican herbicides[END_REF][START_REF] Vavoulidou | Response of soil quality indicators to the pesticide cadusaphos[END_REF]). The endosulfan (insecticide) applied at elevated level (100 ppm) increased significantly arylsulfatase activity. This increase in arylsulfatase activity was transitory and declined with the depletion of applied endosulfan. The short-term effect of endosulfan may be due to its degradation or its gradual adsorption by the soil colloid, making it unavailable for microbes [START_REF] Kalyani | Influence of endosulfan on microbial biomass and soil enzymatic activities of a tropical Alfisol[END_REF]. The same ephemeral effect on arylsulfatase was observed with the fungicide metalaxyl [START_REF] Sukul | Enzymatic activities and microbial biomass in soil as influenced by metalaxyl residues[END_REF]. It is difficult to identify a clear response of this enzymatic activity to pesticides for the same reasons as for urease.

Pattern of variation of enzymatic responses to pesticides

Interactions between pesticides and soil enzymes depend on several factors. Concerning pesticides, persistence, mode of inhibition, toxicity level, concentration and bioavailability are all factors able to influence soil enzyme activities [START_REF] Schaffer | Pesticide effects on enzyme activities in the soil ecosystem[END_REF][START_REF] Gevao | Bound pesticide residues in soils: a review[END_REF]. In the same way, physicochemical and biological soil characteristics, as well as mode of synthesis, expression and inhibition of enzymes are able to modify the expression of soil functions [START_REF] Zimmerman | Organo-Mineral-Enzyme interaction and soil enzyme activity[END_REF]. One way to take into account all these parameters is to realize a global analysis of the literature, with the aim to identify response profiles of enzyme activities subject to pesticide exposure.

In order to identify main patterns of enzymatic responses to addition of pesticides, the previous data were re-analyzed. Thus, based on data of all articles cited in this review, percentages of positive, neutral or negative responses were calculated for each enzymatic activities regardless of pesticide (Table 3). Patterns of responses seem to emerge. Indeed, among hydrolases acid phosphatase, alkaline phosphatase, phosphatases and urease are overall inhibited by pesticides. This inhibition effect is also observed for dehydrogenase activity and is more pronounced whatever pesticides given the number of studies measuring this enzyme. Fluorescein di-acetate hydrolase and arylsulphatase activities are unaffected by pesticide addition. On the contrary, in most cases, cellulase is activated by pesticides and especially by insecticides. The glucosidase is the only enzyme of this analysis that has not an evident response. Both negative and no significant effect is recorded for the activity. The absence of pattern of variation for bglucosidase is probably due to the high redundancy of this activity in microbial communities.

These patterns should be taken with caution due to simplifications needed to reach a generalization; in our case, the variation of pH, field dose and pesticide type were not take account.

A previous review led the author to conclude that the patterns of enzymatic responses were difficult to define in function of pesticide applications. This author had used published results without attempting to standardize their expression in particular the choice of a time step for the observation of the effect or the selection of studies with the closest incubations conditions (here sieved soil). It is therefore consistent to find contradictory results.

Relationships between pesticide mechanisms of action and enzymatic responses

The understanding and interpretation of enzymatic responses after pesticides' addition are very difficult. Indeed, the observed responses are the resultant of numerous factors. There are direct and/or indirect interactions of pesticides with soil enzymes [START_REF] Gianfreda | Interactions between xenobiotics and microbial and enzymatic soil activity[END_REF]. Among them, it can be cited the binding of pesticide with the active site of the enzyme which affect their catalytic activities [START_REF] Tabatabai | Soil enzymes[END_REF] or the use of pesticides as a nutriment source by the microorganisms which may shift not only the balance between the communities but more directly the biosynthesis of enzymes by induction or repression phenomen [START_REF] Cycon | Microbiological characteristics of a loamy sand soil exposed to tebuconazole and l-cyhalothrin under laboratory conditions[END_REF][START_REF] Tejada | Evolution of soil biological properties after addition of glyphosate, diflufenican and glyphosate plus diflufenican herbicides[END_REF][START_REF] Zabaloy | Assessment of microbial community function and structure in soil microcosms exposed to glyphosate[END_REF][START_REF] Chishti | Microbial degradation of chlorpyrifos in liquid media and soil[END_REF]. To these, direct phenomena must also be added the indirect impacts of pesticides on microbial community structure which lead to changes in soil enzymatic activities [START_REF] Bjornlund | Interactions between saprotrophic fungi, bacteria and protozoa on decomposing wheat roots in soil influenced by the fungicide fenpropimorph (Corbel (R)): a field study[END_REF][START_REF] Singh | Microbial degradation of organophosphorus compounds[END_REF][START_REF] Lo | Effect of pesticides on soil microbial community[END_REF]).

These impacts are strongly related to functional redundancy of the target activity [START_REF] Chaer | Comparative resistance and resilience of soil microbial communities and enzyme activities in adjacent native forest and agricultural soils[END_REF][START_REF] Griffiths | Insights into the resistance and resilience of the soil microbial community[END_REF][START_REF] Puglisi | Impact of fungicides on the diversity and function of non-target ammonia-oxidizing micro-organisms residing in a litter soil cover[END_REF]) and the intrinsic properties of soil, pH, humus, clay content or organic matter that influence the accessibility of pesticides [START_REF] Chen | Effects of the fungicides benomyl, captan and chlorothalonil on soil microbial activity and nitrogen dynamics in laboratory incubations[END_REF][START_REF] Gundi | Activities of cellulase and amylase in soils as influenced by insecticide interactions[END_REF][START_REF] Defo | Microcosm study of the long term effect of endosulfan on enzyme and microbial activities on two agricultural soils of Yaounde-Cameroon[END_REF][START_REF] Munoz-Leoz | Non-target effects of three formulated pesticides on microbially-mediated processes in a clay-loam soil[END_REF]. At present, we lack the necessary information on how these different phenomena interact in order to predict a general response for a given enzyme. Moreover, the diversity of experimental conditions (various soils, nature and application rates of pesticides, time after exposure, target enzyme) make comparisons difficult, even if we took the precaution to consider only incubation studies in microcosms under controlled conditions. Finally, the impact of differences between methodological approaches should not be forgotten as reported by [START_REF] Burns | Soil enzymes in a changing environment: current knowledge and future directions[END_REF]. Sample preparation may differ in soil/substrate ratio, pH of the buffer, time and temperature of incubation, soil preparation (sieved or not) and soil storage (fresh, dried or frozen), soil pretreatment (chemicals), assessment of both intra-and extracellular enzymes, or only extracellular enzymes [START_REF] Dick | Soil acid and alkaline phosphatase activity as pH adjustment indicators[END_REF]. Moreover, the assay methods used to measure in situ enzymatic activities could be very different based on Para-nitro phenyl substrates adapted from (Tabataba and Bremner (1970a) or on microplate fluorimetry assays using the fluorescent compound 4-methylumbelliferone [START_REF] Marx | A microplate fluorimetric assay for the study of enzyme diversity in soils[END_REF][START_REF] Pritsch | A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system[END_REF][START_REF] Drouillon | Performance of para-nitrophenyl phosphate and 4methylumbelliferyl phosphate as substrate analogues for phosphomonoesterase in soils with different organic matter content[END_REF][START_REF] Niemi | Stability of the fluorogenic enzyme substrates and pH optima of enzyme activities in different Finnish soils[END_REF][START_REF] Winding | Comparison of CLPP and enzyme activity assay for functional characterization of bacterial soil communities[END_REF][START_REF] Forest | An immunoassay for the assessment of total alternative pathway activity[END_REF][START_REF] Trap | Improved effectiveness and efficiency in measuring soil enzymes as universal soil quality indicators using microplate fluorimetry[END_REF].

Through the analysis of the published works reported in this review, we have tried to underline potential relation-ships between the action mechanisms of pesticides and responses of soil enzymatic activities. To that purpose, the same method as above was applied, i.e., percentages of positive, neutral or negative responses were calculated for each mechanism of action whatever enzymes. Mechanisms of action where the number of experiments was \9 were excluded (Fig. 2). The general trends highlighted and reported in Fig. 2 have been confronted with numerous results from other articles dealing with the impact of pesticides on microbial communities.

Concerning the fungicides, both mechanisms of action presented in Fig. 2 seem to induce an overall negative response of enzymatic activities. Indeed, fungi are responsible for mineralization of organic matter in soil and release of available carbon; hence, their disturbance by fungicides may have a harmful impact on microbial communities and their activities. Incidentally, several authors showed under microcosm experiments that fungicides applications at higher rates than recommended had deleterious effects on fungal populations, while the bacterial populations increased (Monkiedje and Spiteller 2002;[START_REF] Moharram | Effect of two systemic fungicides on cellulose decomposing fungi of tomato plants and on some enzymatic activities[END_REF][START_REF] Strickland | Tebuconazole dissipation and metabolism in Tifton loamy sand during laboratory incubation[END_REF][START_REF] Cycon | Microbiological characteristics of a loamy sand soil exposed to tebuconazole and l-cyhalothrin under laboratory conditions[END_REF][START_REF] Bending | Fungicide impacts on microbial communities in soils with contrasting management histories[END_REF][START_REF] Cycon | Responses of indigenous microorganisms to a fungicidal mixture of mancozeb and dimethomorph added to sandy soils[END_REF]. This switch between fungal and bacterial communities is also explained by the increased levels of nutrients and energy sources released from dead fungal hyphae [START_REF] Cycon | Microbiological characteristics of a loamy sand soil exposed to tebuconazole and l-cyhalothrin under laboratory conditions[END_REF][START_REF] Tejada | Effects of Prochloraz fungicide on soil enzymatic activities and bacterial communities[END_REF]). Moreover, Munoz-Leoz et al. (2011) demonstrated a decrease in microbial biomass in parallel to the decrease in enzymatic activities after fungicide application. This imbalance of microbial populations may lead to the global negative response of enzymatic activities regarding an incubation time range from 28 to 50 days. In field conditions, many authors found the same negative trend on soil enzymes [START_REF] Niewiadomska | Effect of carbendazim, imazetapir and thiramon nitrogenase activity, the number of microorganisms in soil and yield of red clover (Trifolium pretense L)[END_REF][START_REF] Niewiadomska | Pesticide side effect on the symbiotic efficiency and nitrogenase activity of Rhizobiaceae bacteria family[END_REF] even at recommend field rate after only 3 years of fungicide treatments [START_REF] Niemi | Microbial toxicity and impacts on soil enzyme activities of pesticides used in potato cultivation[END_REF].

For the insecticides, our bibliographic analysis showed that the global enzymatic response is different according to the mechanism of action. Insecticides that altered the movement of ions across the nerve cell membranes induce rather a positive response of soil enzymatic activities while insecticides inhibiting the enzyme acetylcholinesterase of nerve impulses caused rather a negative response (Fig. 2). Organochlorine and particularly endosulfan, responsible for global positive responses, are the most commonly and widely used insecticides worldwide. With the application of endosulfan, an increase in microbial biomass carbon is observed [START_REF] Kalyani | Influence of endosulfan on microbial biomass and soil enzymatic activities of a tropical Alfisol[END_REF]. This result may be the consequence of bacterial biomass increase despite fungal biomass may be reduced [START_REF] Joseph | Endosulfan has no adverse effect on soil respiration[END_REF][START_REF] Xie | A short term study on the interaction of bacteria, fungi and endosulfan in soil microcosm[END_REF]). Moreover, whatever the impact of endosulfan on the equilibrium of microbial population, many authors reported the ability of telluric bacteria or fungi to degrade this insecticide which could induce an increase in microbial biomass and/or an activation of enzymatic production [START_REF] Bhalerao | Biodegradation of organochlorine pesticide, endosulfan, by a fungal soil isolate, Aspergillus niger[END_REF][START_REF] Kataoka | Biodegradation of dieldrin by a soil fungus isolated from a soil with annual endosulfan applications[END_REF][START_REF] Kataoka | Biodegradation of endosulfan by Mortieralla sp. strain W8 in soil: influence of different substrates on biodegradation[END_REF][START_REF] Castillo | Isolation of an endosulfan-degrading bacterium from a coffee farm soil: persistence and inhibitory effect on its biological functions[END_REF][START_REF] Yu | Isolation and characterization of an endosulfan degrading strain, Stenotrophomonas sp. LD-6, and its potential in soil bioremediation[END_REF]). On the contrary, insecticides of the organophosphate family create an overall inverse response of enzymatic activities compared to endosulfan. Indeed, chlorpyrifos, which is representative of this insecticide type widely studied, decreased the soil microbial biomass, bacterial, fungal and actinomycetes populations at a concentration corresponding to 20 time the field recommended rate [START_REF] Shan | Effect of chlorpyrifos on soil microbial populations and enzyme activities[END_REF][START_REF] Vischetti | Microbial impact of the pesticide chlorpyrifos on Swedish and Italian biobeds[END_REF]. A same result was found for another molecule, monocrotophos [START_REF] Zayed | Impact of repeated pesticide applications on the binding and release of methyl ( 14 )C-monocrotophos and U-ring labelled ( 14 )C-carba-ryl to soil matrices under field conditions[END_REF], and a field experiment confirmed this alteration of microbial community structure and functions [START_REF] Susan | Effect of organophosphorus insecticides on soil microbial and fungal communities[END_REF]). However, other molecules of organophosphate group had no adverse effects on soil bacterial and fungal counts [START_REF] Martinez-Toledo | Effect of the insecticides methylpyrimifos and chlorpyrifos on soil microflora in an agricultural loam[END_REF][START_REF] Tejada | Evolution of soil biological properties after addition of glyphosate, diflufenican and glyphosate plus diflufenican herbicides[END_REF] or increased slightly these microbial populations [START_REF] Das | Influence of insecticides on microbial transformation of nitrogen and phosphorus in Typic Orchragualf soil[END_REF]. The results of the literature concerning the study of insecticides on microbial communities are consistent with the major trends built from the analysis of 50 articles.

Finally, concerning herbicide effects on soil enzymes based on their mechanism of action show both negative and neutral responses (Fig. 2). Herbicides that inhibit the acetolactate synthase enzyme and photosynthesis process have predominately neutral effect on soil enzymatic activities. The results of the literature are consistent with the observed trends. The addition of atrazine [START_REF] Radivojevic | The impact of atrazine on several biochemical properties of cherno-zem soil[END_REF] or metsulfuron-methyl herbicides [START_REF] Zabaloy | An integrated approach, to evaluate the impacts of the herbicides glyphosate, 2,4-D and metsulfuron-methyl on soil microbial communities in the Pampas region, Argentina[END_REF] induced, respectively, no effect or minor changes to soil microbial activity, bacterial density and functional richness. Effect of long-term atrazine and metolachlor applications at fields demonstrated also that structure of total bacterial community and Acidobacterium, Actinomycetes, methanotroph groups were not severely affected [START_REF] Seghers | Pollution induced community tolerance (PICT) and analysis of 16S rRNA genes to evaluate the long-term effects of herbicides on methanotrophic communities in soil[END_REF].

Herbicides that inhibit the 5-enolpyruvylshikimate-3-phosphate synthase are widely represented by glyphosate. The application of this molecule leads to negative responses of soil enzymatic activity in 77 % of experiments reported in this review (Fig. 2). Studies on abundances and structures of microbial communities of soil or rhizosphere demonstrated that recommended field rate of glyphosate had a benign effect [START_REF] Barriuso | Glyphosate affects the rhizobacterial communities in glyphosate-tolerant cotton[END_REF][START_REF] Hart | Separating the effect of crop from herbicide on soil microbial communities in glyphosateresistant corn[END_REF]) while at a high concentration were observed a short-term stimulation of bacteria [START_REF] Ratcliff | Changes in microbial community structure following herbicide (glyphosate) additions to forest soils[END_REF][START_REF] Weaver | Effects of glyphosate on soil microbial communities and its mineralization in a Mississippi soil[END_REF]). These microbial structure data are in opposition with the results of the studies used to build Fig. 2. In fact, the authors who observed the decrease in enzymatic activities described in parallel an impact on microbial communities. This observation weakens the hypothesis of a potential spread of observations whatever the context.

Conclusion

During last decades, pesticides were increasingly used in agriculture in order to limit crop diseases and increase food production. Today we find increasing amounts of pesticides in the different environmental compartments: water, air and soil. Several investigations have been devoted to study the effect of pesticides on a few parameters of soils. More recently, there have been a number of publications, which recommended soil microcosm, or terrestrial model ecosystem approaches, to assess the effect of pesticides on multiple ecological process. It is difficult to understand the role of pesticides in perturbing the microbial communities and their enzymatic activities in soil due to divergent research findings reported in the literature. A number of factors could be responsible for those controversial results such as soil properties, chemical nature and concentration of pesticides, biological function observed. Even if pesticides applied at recommended rates may cause slight and transient changes to populations or activities of soil microorganisms [START_REF] Johnsen | Pesticide effects on bacterial diversity in agricultural soils: a review[END_REF], it is obvious that long-term recurrent applications of pesticides are known to interfere with the biochemical balance, which can reduce soil fertility and productivity by affecting local metabolism and enzymatic activities. To preserve the environment, many of those molecules have been and will be withdrawn from the market such as clothianidine, imidaclopride, thiame´thoxame and endosulfan. This work has allowed to (i) identify patterns of enzymatic activity response to pesticides' application, (ii) link them with the pesticides' mechanisms of action, (iii) classify the pesticides according to their stimulating, inhibiting or neutral effects on enzymatic activities (iv) bring overall trends face to face with literature related to pesticides impacts on microbial com-munities. Those observations must be considered with care in regard with the number of papers analyzed and the approach used to make conclusions. However, the diversity of contexts and approaches that constitute the basis of this analysis can strengthen our conclusions.

The case of glyphosate weakens the hypothesis of a potential spread of observations whatever the context. Nevertheless, the other general trends described appeared to be confirmed. These first patterns of response have to be validated by further studies which may rely on the development of new technologies such as sensitive molecular-based approaches for measuring microbial community structure (e.g., pyrosequencing), as well as the use of real-time PCR or proteomic approaches to evaluate the expression level of genes involved in key ecological functions. For researchers, the design of experiments related to pesticide effects on soil functioning is difficult especially regarding the choice of the enzymatic activities to be monitored, the pesticides family and rates to be applied, or the intrinsic parameters of soils.

Finally, in this review, we attempt to look for general trends of enzymatic responses to pesticides, which could be useful for researchers and thus for policy decision markers in order to replace agronomy in the center of agriculture.

Fig. 1 .

 1 Fig. 1. Agricultural landscapes and microcosms

Table 1 .

 1 Examples of pesticides used in agricultural systems

Table 2 .

 2 Effect of pesticides on soil enzyme activities in different experimental conditions

Table 3 .

 3 Overall effects of pesticides on enzymatic activities 34


Flora Alarcon

Violaine Planté-Bordeneuve

Gregory Nuel

Study of the Parent-of-origin effect in monogenic diseases with variable age of onset. Application on ATTRv

Keywords: Semi-parametric estimation, Survival Curve, Parent of origin effect, Proportionnal hazard model, Familial amyloid polyneuropathy

In genetic diseases with variable age of onset, an accurate estimation of the survival function for the mutation carriers and also modifying factors effects estimations are important for the management of asymptomatic gene carriers across life. Among the modifying factors, the gender of the parent transmitting the mutation (i.e. the parent-of-origin effect) has been shown to have a significant effect on survival curve estimation on transthyretin familial amyloid polyneuropathy (ATTRv) families. However, as most genotypes are unknown, the parent-of-origin must be calculated through a probability estimated from the pedigree. We propose in this article to extend the method providing mutation carrier survival estimates in order to estimate the parent-of-origin effect. The method is both validated on simulated data and applied to familly samples with 1 ATTRv.

Introduction

In variable age of onset diseases caused by a deleterious gene mutation, knowledge of the survival function for carrier individual is important both to understand the underlying mechanism of the disease (like identification of potential factors that modulate this age of onset) and for prevention strategies. In the literature on this subject, the age-specific cumulative distribution function (CDF), named also penetrance function, is preferentially used. In this paper, like in [START_REF] Alarcon | Nonparametric estimation of survival in age-dependent genetic disease and application to the transthyretin-related hereditary amyloidosis[END_REF],

we will use the classical survival function (which is simply the complementary of the CDF) to assess the probability of not being affected by the disease according to the age for mutation carrier individuals. Note that our survival function hence corresponds to the cause-specific survival (disease diagnosis) and not to the overall survival.

Recently, a semi-parametric method based on a Cox model was developed to estimate survival function from familial data ascertained through affected individual. This method is very efficient and handle the unknown genotypes through the sum-product algorithm in Bayesian network.

The method is described in detail in [START_REF] Alarcon | Nonparametric estimation of survival in age-dependent genetic disease and application to the transthyretin-related hereditary amyloidosis[END_REF]. This method has been applied on families affected by a mendelian disease : the ATTRv, that is the most frequent familial amyloidosis, with autosomal dominant transmission. A fatal outcome occurs after an average duration of 10-13 years [START_REF] Planté-Bordeneuve | Transthyretin related familial amyloid polyneuropathy[END_REF][START_REF] Suhr | Malnutrition and gastrointestinal dysfunction as prognostic factors for survival in familial amyloidotic polyneuropathy[END_REF]. This severe diseases shows important differences in age of onset and thus on survival curve, according to different covariates as countries, gender, mutations [START_REF] Hellman | Heterogeneity of penetrance in familial amyloid polyneuropathy, attr val30met, in the swedish population[END_REF][START_REF] Holmgren | Geographical distribution of TTR met 30 carriers in northern Sweden[END_REF][START_REF] Gorram | New data on the genetic profile and penetrance of hereditary val30met transthyretin amyloidosis in sweden[END_REF]. Especially, heterogeneity in the survival curves according to gender of the transmitting parent have been noted on Portugueses ATTRv families [START_REF] Sousa | Genetic epidemiology of familial amyloidotic polyneuropathy (FAP)-Type I in Povoa do Varzim and Vila do Conde (North of Portugal)[END_REF][START_REF] Ines | Epidemiology of transthyretin familial amyloid polyneuropathy in portugal: a nationwide study[END_REF]. In particular, Hellman et al. [START_REF] Hellman | Heterogeneity of penetrance in familial amyloid polyneuropathy, attr val30met, in the swedish population[END_REF] have find that the risk of disease in the carriers was significantly higher when the mutation was inherited from the mother than from the father.

In 2009, Bonaïti et al [START_REF] Bonaïti | Parent-of-origin effect in transthyretin related amyloid polyneuropathy[END_REF] investigate the parent-of-origin effect in a sample of French and Portuguese families that have already been described in [START_REF] Plante-Bordeneuve | Genetic study of transthyretin amyloid neuropathies: carrier risks among French and Portuguese families[END_REF]. The covariate for parent of origin factor was not calculated algorithmically according to pedigrees and genotypical information but was determined previously to the estimation of the parameters and decided manually by expertbased review. They found that the penetrance was higher when the mutation was inherited from the mother than from the father (i.e. the survival curve was lower when the mutation was inherited from the mother than from the father). This difference was significant in the Portuguese families but not in the French sample. This results led them to the hypothesis of a genetically determined effect through an imprinting phenomenon.

Unfortunately, the gender of the transmitting parent is not known for most of carriers du to the fact that most of individuals are non-genotyped. And we should be skeptical as to the estimates made in this context. However, no simulation studies have been proposed to evaluate the quality of survival curve estimations in studying the parent-of-origin effect.

In this article, we propose to extend the semi-parametric method based on a Cox model described in [START_REF] Alarcon | Nonparametric estimation of survival in age-dependent genetic disease and application to the transthyretin-related hereditary amyloidosis[END_REF] in order to determine the gender of the transmitting parent. The method for analysing the parent-of-origin effect in survival curve estimations of Mendelian disease when a part of genotypic information is unknown, is validated through simulations.

To do the link with the literature about the subject, we illustrate our results on Portuguese and French datasets of ATTRv already analyzed in [START_REF] Bonaïti | Parent-of-origin effect in transthyretin related amyloid polyneuropathy[END_REF] and compare our results with theirs.

Methods

Survival curve are estimated with a semi-parametric method based on a Cox model and adapted for pedigree data. As mentioned before, the data available are families ascertained through at least one affected individual. For all individuals, we have information about his genotype, phenotype, age, gender, etc. Families data consist of individuals independents from each other conditionally to their genotype. The notion of family is taken into account exclusively in the estimation of the probability to carry the mutation. The method is described in detail in [START_REF] Alarcon | Nonparametric estimation of survival in age-dependent genetic disease and application to the transthyretin-related hereditary amyloidosis[END_REF]. In this section, the method is briefly presented in the particular case of studying the parent-of-origin effect.

The model

If we denote by ev the evidence which consists of all the available information: all time T i and status δ i , the individual genotype X i and covariates Z i (possibly multidimensional: gender, comorbidities, ethnicity, etc.), and the genetic testing G i (when available, NA is missing) we have:

P(X, ev) = i P(T i , δ i |X i , Z i )P(G i |X i ) 1 G i =NA φi(Xi) i∈F P (X i ) i / ∈F P X i |X pat i , X mati
where F is the set of founders and where X pat i and X mati indicate respectively the genotype of the father and the mother on individual i. Thus, the genotype distribution among founders follows the Hardy-Weinberg equilibrium with disease allele frequency q, and the conditional distribution for non-founders follows the Mendelian transmission of alleles. If G i = NA, then

X i ∈ {0, 1 m , 1 p , 2}
corresponding respectively to a non-mutated individual, a mutated individual whose mutation comes from the father, a mutated individual whose mutation comes from the mother and finally, to a homozygous individual.

More precisely, we have :

P(T i = t, δ i = 0|X i = x, Z i ) =            exp (-Λ 0 (t) exp(Z i γ)) if x = 1m or x = 2 exp (-Λ 0 (t) exp(β + Z i γ)) if x = 1p 1 if X i = 0
and

P(T i = t, δ i = 1|X i = x, Z i ) =            exp (-Λ 0 (t) exp(Z i γ)) × (λ 0 (t) exp(Z i γ)) if x = 1m or x = 2 exp (-Λ 0 (t) exp(β + Z i γ)) × (λ 0 (t) exp(β + Z i γ)) if x = 1p 0 if X i = 0
which can be simplified as follows :

1 λ 0 (t) P(T i = t, δ i = 1|X i = x, Z i ) =            exp (-Λ 0 (t) exp(Z i γ)) exp(Z i γ) if x = 1m or x = 2 exp (-Λ 0 (t) exp(β + Z i γ)) exp(β + Z i γ) if x = 1p 0 if X i = 0
where λ 0 is the baseline hazard and Λ 0 the baseline cumulative hazard. Moreover, β is the Cox's model parameter, to be estimated, for the parent-of-origin effect and γ the parameter vector corresponding to the other covariates. For the genetic testings, we have a simple model with false positive rate ε and false negative rate η:

P(G i = 1|X i = 0) = 1 -ε P(G i = 0|X i = 0) = 1 -η
Note that, in our case, these rates are probably very small (e.g. ε < 1/100 and η < 1/1000)

EM framework

As X i is either partially observed or not observed, we consider this variable as latent and use a classical Expectation-Maximization algorithm in order to maximize the log-likelihood model in parameter of interest.

Model parameters are: q, β, γ, ε, η, Λ 0 and λ 0 . In order for the model to be identifiable, we assume that error rates ε and η are known as well as the disease allele frequency q. And since λ 0 appears only has a proportional factor in the expression of φ i (X i ) we can perform model inference without explicit value for this parameter. Our aim is therefore to estimate θ = (β, γ, Λ 0 ) using a classical EM framework.

In this framework we alternate to steps until convergence: E-Step give the weights w i using current parameter θ old , computed for all i:

w pat i = P (X i = 1p|ev; θ old ) and w mat i = P (X i = 1m or X i = 2|ev; θ old ) M-

Posterior distributions

For computing the posterior weights w pat i and w mat i of the E-Step one has to integrate the model over all unobserved (even when partially observed) genotypes X i . Since the total number of configurations for X i is 4 n it is clearly impossible to perform this summation with brute force.

Fortunately, geneticist know for year that likelihood computations in genetic model can be performed efficiently using the Elston-Stewart algorithm [START_REF] Elston | A general model for the genetic analysis of pedigree data[END_REF]. Thanks to this algorithm, it is therefore possible to compute any posterior distribution as a simple likelihood ratio:

P(X i = x|ev) = P(X i = x, ev) P(ev)
In the probabilistic graphical model community, such computation can be dealt efficiently using the sum-product algorithm (also called: belief propagation, forward/backward, inward/outward, Kalman filter, etc.) in order to obtain in a single computational pass all w pat i and w mat i . NB:

in Totir [START_REF] Liviu R Totir | An efficient algorithm to compute marginal posterior genotype probabilities for every member of a pedigree with loops[END_REF] , the authors suggest a generalization of the Elston-Stewart algorithm allowing to compute all posterior distribution in a single passe rather than repeating likelhood computations.

Practically, initialization is performed by affecting random weights w i (ex: drawn from a uniform distribution on [0, 1]). EM iterations are stopped when we observe convergence on test survival estimates (ex: baseline survival at age 20, 40, 60, 80).

Results

Simulations study

We have simulated n families (n = 100 or 400) with 10 individuals as shown in figure 1. transmitted by the father. The disease allele frequency was set to q = 0.20 in our simulated dataset for the sake of speed (without simulating any ascertainment process). The age at event genotypes are observed, the algorithm converges in 65 iterations on average while it converges in 89 iterations on average when no genotypes are observed. We also observe a higher variability of the number of iterations when the genotypes are not observed.

q q q q -2 -1 0 1 S0 S1 S2 Oracle scenario estimation A q q q q -1.5 -1.0 -0.5 0.0 0.5 S0 S1 S2 Oracle scenario estimation B q q q q -8 -4 0 S0 S1 S2 Oracle

Application

Our method has been applied to two datasets of Portuguese families and French families already analyzed in [START_REF] Bonaïti | Parent-of-origin effect in transthyretin related amyloid polyneuropathy[END_REF]. For data analysis, the disease allele frequency was set to q = 0.04 as in [START_REF] Hellman | Heterogeneity of penetrance in familial amyloid polyneuropathy, attr val30met, in the swedish population[END_REF].

The ascertainment bias was corrected by a classical method that consists in simply removing the phenotypic information of the proband, as done in [START_REF] Alarcon | Nonparametric estimation of survival in age-dependent genetic disease and application to the transthyretin-related hereditary amyloidosis[END_REF]. The parent of origin gender parameter was not significantly different from zero in French families ( β = 0.114 with p-value = 0.467).

q q q q q q q q q q q q q q q q q q q q q q q q q q 0 100 200 300 400 500 S0 S1 S2 scenario iteration_number A q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q q 0 200 400 ) showing that the risk of being affected is higher when the mutation is transmitted by the mother than by the father. This difference in survival curves estimated is shown in Figure 4 and is totally consistent with the results of [START_REF] Bonaïti | Parent-of-origin effect in transthyretin related amyloid polyneuropathy[END_REF].

S0

Conclusion

In this article, we propose an extension of our method in order to take into account the gender of the transmitting parent in the estimation of the survival function from familial data in cases of age-dependent genetic diseases. The probability of the transmitting parent gender is calculated and its effect is estimated through a proportional hazard model. Our extension is assessed with simulations and applied on a French and a Portuguese dataset of ATTRv families. Obviously, the method provides confidence intervals for each estimation.

The simulation study shows that the method is unbiased and the increase in variance of the We have applied this method to a French and Portuguese dataset and shows a significant difference of the penetrance function according to the gender of the transmitted parent. These results are consistent with the analyses already done in [START_REF] Bonaïti | Parent-of-origin effect in transthyretin related amyloid polyneuropathy[END_REF], even if the method used in [START_REF] Bonaïti | Parent-of-origin effect in transthyretin related amyloid polyneuropathy[END_REF] was less precise since the genotypic probabilities were not calculated. Results are also consistent with recent works in the Portuguese and Swedish ATTRV30M families showing that the disease's risk and anticipation in the age of onset increase among offspring of affected mothers [START_REF] Lemos | Overcoming artefact: anticipation in 284 portuguese kindreds with familial amyloid polyneuropathy (fap) attrv30m[END_REF][START_REF] Gorram | New data on the genetic profile and penetrance of hereditary val30met transthyretin amyloidosis in sweden[END_REF]. The hypothesis of a role for mithochondrial DNA could explain this increased risk when the mutation is transmitted by the mother. The advantage here is that we have proposed an unified method able to take into account precisely these probabilities. Moreover, the method allows additional variables to be taken into account when estimating survival curves.

Finally, in this work, genotypic probabilities are calculated using a sum-product algorithm which is a very general method which can deal efficiently with very complex pedigree structure (ex: 2000 individuals with 50 loops). Unlike Elston-Stewart algorithm, the sum-product algorithm does not use loop breaking approaches to deal with loop pedigrees. Instead, the sumproduct algorithm use an auxiliary tree called the junction tree (JT) which basically is a clique decomposition of the moral graph corresponding to the pedigree problem. JT and BP are well known is the graph theory (ex: JT can be used to solve a graph coloring problem) and in the mathematical field of probabilistic graphical models (Bayesian network, hidden Markov model, decision trees, Markov networks, etc.).

Figure 1 :

 1 Figure 1: Simulated family structure

Figure 2 :

 2 Figure 2: Violin Plots according to different scenarios. A : n = 100 families ; β = -0.6. B : n = 400 families ; β = -0.6. C : n = 100 families ; β = -1.2

Figure 3 :

 3 Figure 3: Violin plot with box plot of the number of iterations before EM convergence according to different scenarios. A : n = 100 families ; β = -0.6. C : n = 100 families ; β = -1.2

Figure 4 :

 4 Figure 4: Estimation of the survival function according to the sex of the transmitting parent with 95% confidence intervals for Portugese families

  Step : create an artificial weighted dataset with the following 2n patients

	time status POO covariates weight
	T 1	δ 1	pat	Z 1	w pat 1
	. . .	. . .	. . .	. . .	. . .
	T n	δ n	pat	Z n	w pat n
	T 1	δ 1	mat	Z 1	w mat 1
	. . .	. . .	. . .	. . .	. . .
	T n	δ n	mat	Z n	w mat n
	and simply:

• fit a (weighted) Cox's proportional hazard model with Factor POO and the covariates to update β and γ

• use non-parametric estimate (e.g. Kaplan-Meier or similar) of Λ 0 .

was simulated according to a piecewise constant hazard rate function. Moreover a censoring variable was added that follows a uniform distribution U[15, 80].

To study the parent-of-origin effect, the POO was simulated in a proportional hasard model.

The risk when the mutation is transmitted by the mother is given by λ 0 as follows and the coefficient parameters in the hazard model is noted β (β = -0.60 or β = -1.2 in simulations). Thus, as β < 0, that's mean that survival is upper when mutation provides on the father.

To study the behavior of our method, we will consider different scenarios for simulations. In the first scenario (S0), all genotypes are set to unobserved, in the second more realistic scenario (S1), 80% of genotypes are observed in affected individuals and only 10% are observed in non affected individuals and in the third one (S2), all genotypes are observed. Moreover, we consider an other scenario where, in addition to the genotypes, we know, for each individual, the sex of the parent transmitting the mutation. We call this scenario "Oracle". In order to compare the β estimate in the different scenarios, 200 replications are performed.


Laurent O Martinez
email: laurent.martinez@inserm.fr

Bertrand Perret

Annelise Genoux

Update on PCSK9 inhibitors, lipoprotein(a) and cardiovascular risk

This recent meta-analysis based on 41 randomized-controlled trials comparing PCSK9 inhibitors (alirocumab or evolocumab) to placebo and/or ezetimibe and/or other lipid-lowering therapy shows that PCSK9 inhibitors reduced Lp(a) levels by -26.7% (95% CI, -29.5% to -23.9%) with a significant heterogeneity within studies according to comparator and duration of treatment.

19.

Lipoprotein(a) STRUCTURE AND ASSOCIATED CARDIOVASCULAR RISK

Lipoprotein (a) (Lp(a)) is constituted of an low-density lipoprotein (LDL)-particle whose apoB is covalently linked to apo(a). The latter contains several 'kringle' domains analogous to those of plasminogen. Moreover, the number of kringle IV type 2 repeats is variable, under genetic control, and inversely related to the plasma concentration of Lp(a) [START_REF] Wilson | Use of Lipoprotein(a) in clinical practice: A biomarker whose time has come. A scientific statement from the National Lipid Association[END_REF]. An increased risk of coronary artery disease (CAD) has been reported in carriers of LPA genetic variants, associated with elevated Lp(a) and reduced kringle IV-2 repeats [START_REF] Clarke | Genetic Variants Associated with Lp(a) Lipoprotein Level and Coronary Disease[END_REF]. In general, population-based prospective studies have demonstrated a continuous association of Lp(a) concentration with the risk of CAD and ischemic stroke [START_REF] Erqou | Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality[END_REF]. Likewise, in patients with established cardiovascular (CV) disease treated with statins, higher levels of Lp(a) were associated with further CV events, independently of LDL-cholesterol (LDL-C) [4**,5]. A 20 mg/dL greater Lp(a) would be associated with an 8% increase in CV risk [START_REF] Wong | Relation of First and Total Recurrent Atherosclerotic Cardiovascular Disease Events to Increased Lipoprotein(a) Levels Among Statin Treated Adults With Cardiovascular Disease[END_REF][START_REF] Arnold | Lipoprotein(a) is associated with large artery atherosclerosis stroke aetiology and stroke recurrence among patients below the age of 60 years: results from the BIOSIGNAL study[END_REF]. Conversely, in patients on secondary prevention, lowering Lp(a) levels by 50mg/dl should reduce CV diseases by 20% [7**].

Several mechanisms have been proposed to explain the effects of Lp(a) on atherothrombosis. Due to its homology with plasminogen, Lp(a) can in-vitro inhibit fibrinolysis. This might favor thrombosis at vulnerable arterial plaques [START_REF] Wilson | Use of Lipoprotein(a) in clinical practice: A biomarker whose time has come. A scientific statement from the National Lipid Association[END_REF]. Besides, Lp(a) has long been described in human coronary atheroma lesions, colocalized with macrophages. These cells might participate to Lp(a) clearance, since Lp(a) is only partly catabolized through the LDL receptor (LDL-R).

Recently, elevated Lp(a) and oxidized phospholipids (ox-PL) were found associated with more prevalent aortic valve stenosis (AVS) and microcalcification, documented by positron emission tomography [8*]. In patients with moderate AVS, elevated Lp(a) and ox-PL conferred greater progression of valve calcification and increased risk of valve replacement or cardiac death [9,10**]. Similar observations were made in hypercholesterolemic patients [START_REF] De Isla | Lipoprotein(a), LDL-cholesterol, and hypertension: predictors of the need for aortic valve replacement in familial hypercholesterolaemia[END_REF]. Ox-PL and Lp(a) are linked in arterial and aortic valve lesions. This complex might promote lipid deposition, endothelial dysfunction, inflammation, osteogenic differentiation and calcification [10**].

PROPROTEIN CONVERTASE SUBTILISIN/ KEXIN TYPE 9 AND ANTI-PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9

Proprotein convertase subtilisin/kexin type 9 structure, functions and association to cardiovascular risk.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory serine protease mainly expressed in the liver and to a lesser extent in kidney, lung, intestine, central nervous system, inflammatory and blood vessel cells. The PCSK9 gene encodes for a 72 kDa inactive protein (pro-PCSK9) containing a signal peptide. Pro-PCSK9 is cleaved to form the mature 62 kDa PCSK9 protein. The PCSK9 gene expression is regulated by different transcription factors among which sterolresponse element-binding protein 2 (SREBP2) which also regulates LDL-R expression [12,13*].

PCSK9 was first discovered as a genetic determinant of some forms of familial hypercholesterolemia (FH). In approximately 1% of cases, FH with severely elevated LDL-C levels results from the presence of 'gain-of-function' mutations on PCSK9. Conversely, PCSK9 loss-offunction mutations are associated with very low plasma levels of LDL and to a lesser CV risk [14]. High circulating PCSK9 is significantly associated with increased risk of major adverse cardiovascular events (MACE) [15*]. PCSK9 binds to the LDL-R at the surface of hepatocytes and prevents its recycling after endocytosis, leading to its lysosomal degradation. Overexpression of PCSK9 results in a reduced clearance and an accumulation of LDL in the circulation [13*]. Recent findings demonstrate that PCSK9 is also involved in the pathophysiology of atherosclerosis, independently from LDL-R cleavage, by actively modulating inflammation, plaque formation and thrombosis [13*,16].

Proprotein convertase subtilisin/kexin type 9 inhibitors, effects on the cardiovascular risk

Given its established role as a major LDL-R inhibitor, PCSK9 has become a prime therapeutic target to lower LDL-C. Currently, two main approaches are used to decrease circulating PCSK9: binding of PCSK9 with human monoclonal antibodies (alirocumab or evolocumab) or inhibiting its hepatic synthesis with a small interfering RNA (inclisiran). With any of these strategies, PCSK9 inhibitors (PCSK9i) impair LDL-R degradation and lower LDL-C by 50 -60%. Consequently, they reduce the risk of MACE when added to statins [START_REF] Steffens | PCSK9 inhibitors and cardiovascular outcomes[END_REF]. predicted the risk reduction of a first primary outcome event (CV deaths, nonfatal MI, unstable angina or ischemic stroke) [START_REF] Bittner | Effect of Alirocumab on Lipoprotein(a) and Cardiovascular Risk After Acute Coronary Syndrome[END_REF], of a first major peripheral artery disease [START_REF] Schwartz | Peripheral Artery Disease and Venous Thromboembolic Events after Acute Coronary Syndrome: Role of Lipoprotein(a) and Modification by Alirocumab: Prespecified Analysis of the ODYSSEY OUTCOMES Randomized Clinical Trial[END_REF] and also of recurrent events [25**]. These clinical trials were conducted in a secondary prevention setting, and primary prevention trials regarding the protective effects of Lp(a) reduction are still lacking. Moreover, it remains difficult to dissociate the protective effects of PCSK9i through LDL-C lowering and/or Lp(a) lowering. A treatment that selectively lowers Lp(a) should provide a real proof of causality between Lp(a) lowering and CV risk reduction. An antisense oligonucleotide (pelacarsen) targeting apo(a) mRNA reduces mean Lp(a) levels by 80%. It is currently under evaluation in the phase 3 HORIZON (Assessing the Impact of Lipoprotein(a) Lowering With TQJ230 on Major Cardiovascular Events in Patients With CVD) trial to assess the impact of Lp(a) lowering on MACE in patients with CV disease [START_REF] Tsimikas | Emerging RNA Therapeutics to Lower Blood Levels of Lp(a): JACC Focus Seminar 2/4[END_REF].

Proprotein convertase subtilisin/kexin type 9, proprotein convertase subtilisin/kexin type 9 inhibitors and calcified aortic stenosis

Besides Lp(a), PCSK9 itself had been found in human aortic valves and may participate to calcium deposition [START_REF] Poggio | PCSK9 Involvement in Aortic Valve Calcification[END_REF]. Incubation of valve interstitial cells in an osteogenic medium stimulates PCSK9 synthesis and secretion, concomitant with a rise in intracellular calcium. This latter effect is blocked by a neutralizing anti-PCSK9 antibody [30**]. In a large meta-analysis [30**], the common PCSK9 (R46L) loss of function variant has been associated with a 20% lower risk of calcified AVS. In the prospective FOURIER trial, evolocumab reduced aortic stenosis events by 50% beyond one year treatment [START_REF] Agstam | Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors in aortic stenosis -Is this the light at the end of the tunnel for patients with aortic stenosis?[END_REF].

Beyond low-density lipoprotein cholesterol, effects of proprotein convertase subtilisin/ kexin type 9 inhibitors on plasma lipoproteins

The impact of PCSK9i on the lipoprotein profile was recently determined by nuclear magnetic resonance [START_REF] Zhang | Metabolic effects of PCSK9 inhibition with Evolocumab in subjects with elevated Lp(a)[END_REF]. Evolocumab induced a decrease of small and medium-sized LDL particles, potentially associated with a reduction of CV risk. A decrease of all subtypes of very-low density lipoprotein was also observed, which may reflect an accelerated catabolism of those particles [START_REF] Zhang | Metabolic effects of PCSK9 inhibition with Evolocumab in subjects with elevated Lp(a)[END_REF]. In a recent study, anti-PCSK9 antibodies led to a more atheroprotective high-density lipoprotein (HDL) profile, with a rise in total and medium HDL particles, and a decrease of large HDL [33*]. Furthermore, under PCSKi, medium and small HDL were found enriched in esterified cholesterol but depleted in triglycerides, which might result from a reduced cholesteryl ester transfer protein activity due to lower availability of apoB-containing lipoproteins [33*]. Interestingly, small and medium-sized HDL particles appear as preferred acceptors of excess cell cholesterol.

  Unexpectedly and unlike statins, PCSK9i lower plasma Lp(a) levels by 27% on average [18*]. The mechanisms involved in this effect of PCSK9i remain unclear, although several hypotheses been proposed: an enhanced clearance of Lp(a) through the LDL-R, an enhanced Lp(a) clearance via other receptors (LDL-R related protein 1, cluster of differentiation 36 receptor, toll-like receptor 2, scavenger receptor-BI and plasminogen receptors) and a reduction of apo(a) production or of Lp(a) assembly [19]. A high frequency of discordant LDL-C/Lp(a) responses to alirocumab was reported, suggesting that this PCSK9i reduces plasma Lp(a) through alternative mechanisms to the LDL-R [20*]. In the same way, recent kinetic studies using stable isotopes tracer methodologies have shown that PCSK9i do not modulate hepatic uptake of Lp(a) by LDL-R [21*,22], except when combined with statins [21*,23]. In contrast, PCSKi reduce Lp(a) levels by lowering apo(a) production rate [21*,24]. In the FOURIER (Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk) trial, evolocumab significantly reduced the risk of CV death, myocardial infarction (MI) or urgent revascularization in patients with established CV disease. The risk reduction was greater in patients with higher baseline Lp(a) [4**]. In the ODYSSEY OUTCOMES (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab) trial, in patients with the recent acute coronary syndrome, Lp(a) lowering by alirocumab reduced the total burden of CV events independently of LDL-C decrease. Each 5-mg/dL reduction in Lp(a) predicted a 2.5% relative reduction in CV events [25**]. Alirocumab-induced decrease of Lp(a)

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