Kalpana Shrivastava
email: kalpana.shrivastava@uab.cat

Anju Bansal

Bhagwat Singh

Mustoori Sairam

Govindaswamy Ilavazhagan

Ilavazhagan Govindaswamy

Sub-chronic oral toxicity study in Sprague-Dawley rats with hypoxia mimetic cobalt chloride towards the development of promising neutraceutical for oxygen deprivation

Keywords: hypoxia mimetic, Sprague-Dawley, sub-chronic, CoCl 2

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Sub-chronic oral toxicity study in Sprague-Dawley rats with hypoxia mimetic cobalt chloride towards the development of promising neutraceutical for oxygen deprivation

Introduction

Oxygen is the central fulcrum around which life of an aerobic organism rotates. Any deficiency in oxygen concentration (hypoxia) in the body is sufficient to cause an impairment of function. Hypoxia contributes significantly to the pathophysiology of major categories of disease, including myocardial and cerebral ischemia and high altitude related maladies. In the recent years a lot of attention has been focused on high altitude biology and medicine. About 140 million people live permanently at altitudes higher than 2500 m (WHO, 1996). Many people travel to high altitudes for recreational pursuits such as mountaineering, trekking, and skiing. In some countries, the army also deploys thousands of military personnel at high altitude areas to defend the country borders. Moreover, research on high-altitudes also contributes to understanding the processes of adaptation to the environment and evolution [START_REF] Beall | Tibetan, and Ethiopian patterns of adaptation to high-altitude hypoxia[END_REF].

Preconditioning can be defined as a process by which a tissue is rendered more tolerant to a subsequent lethal insult. It is believed to produce intracellular adaptations mediated by hypoxia inducible A c c e p t e d m a n u s c r i p t 3 factor 1α (HIF1α), which is a master regulator of genes activated by low oxygen tension and thus enhance endogenous defense mechanisms [START_REF] Gleadle | Hypoxia and the regulation of gene expression[END_REF]. Hypoxic preconditioning can be mimicked by chemical preconditioning with cobalt chloride (CoCl 2 ) which activates hypoxia-mediated signaling pathways aberrantly under normoxia by stabilizing the cytosolic HIF1α [START_REF] Hirsila | Effect of desferrioxamine and metals on the hydroxylases in the oxygen sensing pathway[END_REF][START_REF] Ho | Effects of transition metals on the expression of the erythropoietin gene: Further evidence that the oxygen sensor is a heme protein[END_REF]Wang et al, 1995). Chemical preconditioning with CoCl 2 has several advantages over physical/environmental preconditioning : 1) number of people that can be preconditioned is not limited as compared to that of physical preconditioning in simulation chambers 2) easy to implement, and 3) economical.

Chemical preconditioning by cobalt has prospective clinical efficacy in a wide variety of pathophysiological processes. Administration of CoCl 2 was shown to provide protection against ischemia reperfusion injury in brain [START_REF] Bergeron | Role of hypoxia inducible factor-1 in hypoxia induced ischemia tolerance in neonatal rat brain[END_REF][START_REF] Miller | Cerebral protection by hypoxic preconditioning in a murine model of focal ischemia-reperfusion[END_REF]. It has been reported that pretreatment with a low dose of CoCl 2 in mice induced cardiac preconditioning (Xi et al, 2004). [START_REF] Matsumoto | Induction of renoprotective gene expression by cobalt ameliorates ischemic injury of the kidney in rats[END_REF] reported that administration of CoCl 2 resulted in a marked protection against ischemic renal injury.

Moreover, it is interesting to consider the finding that the responsiveness of HIF-1 to hypoxia, but not CoCl 2 wanes with age [START_REF] Chavez | Hypoxia inducible factor-1α accumulation in rat brain in response to hypoxia and ischemia is attenuated during aging[END_REF].

As excessive exposure to cobalt is associated with several conditions, including asthma, pneumonia, and hematological abnormalities [START_REF] Lauwerys | Health risks associated with cobalt exposure-an overview[END_REF], and there is large anomaly on the toxicity of CoCl 2 , optimization of CoCl 2 administration protocol and its toxicity studies are of critical importance in studies involving clinical applications. Recently, we have revealed the neuroprotective effect of oral supplementation of CoCl 2 (12.5 mg cobalt kg -1 BW for seven days) on hypobaric hypoxia induced oxidative stress (Shrivastava et al, 2008c) and its efficiency in facilitating acclimatization to hypobaric hypoxia (Shrivastava et al, 2008a). Moreover, we have also studied the efficacy of cobalt preconditioning in preventing hypobaric hypoxia induced vascular leakage (Shrivastava et al, 2008b). Keeping in view the beneficial effects of CoCl 2 in animals and to develop efficacious therapeutic regimen in humans; its toxicity was evaluated in the present study with respect to various hematological, biochemical and histological variables in rats. , 1998). All the studies were approved by the ethics committee of this institute (DIPAS). Efforts were made to minimize animal suffering and number of animals for experimentation purpose.

Materials and methods

Test article

Study design

CoCl 2 at the concentration 12.5 mg cobalt kg -1 BW (orally via gastric cannula, daily once, for 7 days), which we have standardized earlier (Shrivastava et al, 2008a) was tested for its toxicity. Cage side clinical observations were performed each morning on all the animals with an additional afternoon check.

Body weight was recorded everyday from Day 0 till the day of sacrifice. Food and water consumption was assessed on a daily basis. The rats were randomly divided into 4 experimental groups of 8 animals each (Figure 1):

(i) Control: sacrificed after (a) 7 days and (b) 14 days of start of experiment (ii) Group sacrificed after 1hr of 7days CoCl 2 supplementation schedule (iii) Group sacrificed after 24hrs of 7days CoCl 2 supplementation schedule (iv) Group sacrificed after 7 days of 7days CoCl 2 supplementation schedule A complete battery of hematological, biochemical and histological variables were studied at baseline and after end of the schedule. Group 1 (b) control was used for analyzing significant differences in body weight, feed and water intake. Rest of the parameters (hematological, biochemical or histological)

were not significantly different in Group 1 (a) and (b) controls, hence, Group 1(a) was used as standard control for significance analysis.

Sample collection

A c c e p t e d m a n u s c r i p t 5 Blood was collected from all the rats after retro-orbital puncture. A part of blood was added to heparinized tubes and plasma was used for hematological analysis. Another part was used to separate serum for biochemical analysis. The rats were later sacrificed under ketamine hydrochloride (80 mg/kg) anesthesia (Intraperitoneal; i.p.) and various tissues were dissected out after thorough perfusion with normal saline (0.9% NaCl) and stored at -80 0 C for cobalt estimation and in 10% formalin for histology.

Survival, body weight, feed and water intake

Survival was monitored by visual observation and body weight was taken on weighing balance.

Food consumption was assessed on a daily basis by weighing the feeders and expressed as grams per rat per day. Water consumption was measured volumetrically and expressed as ml of water consumed per animal per day.

Hematological analysis

Blood collected from the retro-orbital plexus under light ether anesthesia at specified time intervals was used for various hematological analyses (white blood corpuscles, red blood corpuscles, lymphocytes, granulocytes, monocytes, mean cell volume, mean corpuscular hemoglobin concentration, hematocrit, hemoglobin, and platelets). The analyses were carried out using MS4 autoanalyser (Desing Lab.).

Organ-body weight index (OBI)

Brain, lung, liver, spleen, heart and kidney were dissected soon after perfusion from each group of animals and were weighed immediately. OBI was calculated as: organ weight x 100/animal body weight.

Cobalt metal estimation

Cobalt was estimated in various organs (brain, lung, liver, heart and kidney) using Induced

Coupled Plasma-Atomic absorption Spectrometer (ICP-AAS; Perkin-Elmer). Cobalt standard stock solution was prepared by dissolving 1.000 g of cobalt metal, accurately weighed to at least four significant figures, in a minimum amount of (1:1) 10% HNO 3 and several dilutions were prepared from this stock solution. These dilutions were injected and atomized in the Spectrometer to plot a standard curve from these readings. Later, 100mg of each tissue were dried and charred to ashes in graphite muffle furnace and a homogenous solution was then prepared in 10% HNO 3 to be injected and atomized in the Spectrometer.

Biochemical analysis

Biochemical parameters viz. serum glutamic-pyruvic transaminase (SGPT), serum glutamicoxaloacetic transaminase (SGOT), Creatinine, Creatine kinase, Glucose, LDH and uric acid were measured using RANDOX kits following manufacturer's instructions.

Lipid peroxidation

Lipid peroxidation was assessed in serum by measuring malondialdehyde (MDA) levels i.e.

thiobarbituric acid reactive substances (TBARS) using method of [START_REF] Ohkawa | Assay of lipid peroxides in animal tissues by TBA reaction[END_REF]. Thiobarbituric acid (TBA) was used as a standard, and the levels of lipid peroxides were expressed as nmol MDA/mg protein.

Briefly, 0.5 ml serum was mixed with 1.5 ml of 20% trichloroacetic acid and 0.67% TBA. The mixture was incubated at 80ºC in water bath for 45 min. The samples were then cooled to room temperature, centrifuged for 5min at 5000 rpm and absorbance of the supernatant was measured at 531nm.

Protein estimations

The protein content in the serum was determined using bovine serum albumin as standard [START_REF] Lowry | Protein measurement with Folin Phenol reagent[END_REF].

Histology and Neurotoxicity

Various tissues fixed in 10% buffered formalin were dehydrated in graded series of alcohol (50%, 70%, 90%, 100%) followed by xylene and embedded in paraffin wax. Multiple sections from each block were prepared at 5 µm thickness and stained with hematoxylin and eosin (McManus and Mowry, 1965).

Screening for neurotoxicity was also included in the study protocol. This subjective screening for neurotoxicity consisted of daily cage-side clinical observations and objective gross histopathological analysis of brain tissue and a functional observational battery (FOB) conducted during 7 days on all 32 rats.

Statistical analysis

The results are expressed as mean ±SD. The body weight data were analyzed by one way ANOVA

Results

Survival, Feed-water intake and change in body weight

Mean feed consumption over the duration of the study was comparable across all study groups (Table 1) though daily consumption values did differ sporadically between different treatment groups.

Mean feed intake were also comparable across all study groups. Mean water consumption values are also shown in Table 1. Water intake was increased with time and increase in body weight but was comparable to control. No death was reported during the whole study.

Hematological analysis

Hematological analysis of blood samples is summarized in Table 2. A significant increase (P<0.05) was observed in hemoglobin, hematocrit and RBC after cobalt supplementation and the values were maintained at the higher levels even 7 days after the withdrawal of cobalt supplementation (group 2, 3 and 4). There was a significant increase in monocytes, Granulocytes and WBC observed 1hr and 24hrs after cobalt supplementation (group 2 and group 3 respectively). The values gradually subsided till 7 days following cessation of cobalt supplementation (group 4).

Organ-body weight index (OBI)

Results of OBI are summarized in Table 3. No significant changes in OBI were observed except in heart, liver and lungs (group 2/3) but were comparable with control values after 7 days.

Cobalt metal estimation

Cobalt content analyzed from various organs as brain, lung, liver, heart and kidney is summarized in Table 4. The standard curve had a correlation coefficient of 0.985. The metal content increased significantly in liver (group 2 and 3) but was comparable to control after 7 days (group 4).

Biochemical analysis

There was no significant change in SGPT, SGOT, Creatinine, Creatinine kinase, LDH and uric acid in any of the groups. There was a significant elevation in glucose levels in Group-2 and Group-3 which came back to nearby control values by 7 days of cessation of cobalt supplementation (Group 4).

Results are summarized in Table 5.

A c c e p t e d m a n u s c r i p t 8 Lipid peroxidation and protein estimations

Lipid peroxidation and protein levels showed no significant changes in all the four groups studied.

Refer (Table 6).

Histology and Neurotoxicity

Histology of brain, lung, liver, spleen, heart and kidney did not show any abnormalities produced by cobalt exposure when observed 1h, 24h and 7 days after exposure (figure not shown). Though there was an initial increase in weight and cobalt accumulation in liver (group 2 and 3) but histolopathological analysis rule out liver toxicity. Ophthalmoscopic examination of control and cobalt treated animals during 7 days of the study revealed no treatment-related abnormalities.

Discussion

Cobalt has been proposed to be used as a drug or neutraceutical for human benefit at high altitudes and in conditions involving similar pathophysiology. Though cobalt is the integral part of an essential vitamin i.e. cobalamine or vitamin B12, medicinal use of cobalt has not been previously addressed or supported by FDA. The present study was conducted to address the questions of safety that must be taken care before any substance has FDA approval.

The toxicity of cobalt is very ambiguous and is reported to be dependent on route of administration, pre-existing cardiac problems, age, period of exposure, nutritional status (ATSDR, 2001).

Cobalt (0.16-1mg Co/kg) has been used as a treatment for anemia in pregnant women and infants [START_REF] Holly | Studies on iron and cobalt metabolism[END_REF]. Other beneficial effects include protective responses in liver, heart and renal injuries [START_REF] Kato | Heme oxygenase-1 overexpression protects rat livers from ischemia/reperfusion injury with extended cold preservation[END_REF][START_REF] Kotori | Heme oxygenase -1 overexpression protects rat hearts from cold ischemia/reperfusion injury via an antiapoptotic pathway[END_REF]Tullius et al, 2002). We have earlier reported that supplementation of CoCl 2 (12.5 mg cobalt kg -1 BW, 7days) promotes tolerance and facilitates acclimatization to hypobaric hypoxia (Shrivastava et al, 2008a). It can attenuate hypobaric hypoxia induced oxidative stress (Shrivastava et al, 2008c) and vascular leakage in brain (Shrivastava et al, 2008b). The possibility of use of cobalt either in the form of a drug or neutraceutical prompted us to evaluate the safety of this standardized dose (12.5 mg cobalt kg -1 BW, 7days).

In the present study the sub-chronic toxicity of an effective oral dose of 12.5 mg cobalt kg -1 BW has been evaluated; based on various hematological and biochemical parameters in rats. The metal content was also assessed in various tissues indicating negligible adverse effect of any kind. Most of the effects that carrying capacity of the blood (Shrivastava et al, 2008a). This effect was persistent even after a week (group 4) of cobalt supplementation.

Another important parameter is blood glucose concentration as glucose is the only nutrient that can be used by the brain, retina, and germinal epithelium of the gonads in sufficient quantity to supply them optimally with their required energy. Moreover, blood glucose is a measure of normal liver function as it functions as an important blood glucose buffer system. In the present study a significant increase in glucose concentration was observed in the blood after cobalt supplementation but was well within normal range. A significant increase in blood glucose was also noted in our previous study after 7 days of cobalt supplementation due to an increase in Glut-1 levels (Shrivastava et al, 2008a). The level of glucose returned to near control value after 7 days.

Liver injury was evident in patients with beer-cobalt cardiomyopathy characterized by increased levels of serum bilirubin, SGPT, SGOT, LDH [START_REF] Alexander | Cobalt-beer cardiomyopathy. A clinical and pathologic study of twenty-eight cases[END_REF][START_REF] Morin | Cobalt Cardiomyopathy: Clinical Aspects[END_REF]. Following this SGPT, SGOT, LDH levels were estimated in blood-serum of animals along with the liver weight. No significant change in these enzymes indicates normal liver function but there was some increase in liver weight which was later normalized. These results are in accordance with previous reports showing no morphological or enzymatic changes in livers of rats supplemented with 2.5-30.2 mg Co/Kg for 3-7 months [START_REF] Domingo | Cobalt in the environment and its toxicological implications[END_REF][START_REF] Krasovskii | Experimental data for substantiating the maximum permissible concentration of water reservoirs[END_REF].

Other biochemical parameters estimated showed no significant changes viz., creatinine (kidney function test), creatine kinase (marker for skeletal muscle disease, myocardial infarction and cerebrovascular accidents), uric acid and protein. No significant changes were seen in lipid peroxidation, also OBI for vital organs and hematological parameters at 12.5 mg Co/Kg BW did not show any significant changes. Cobalt concentration in tissues are generally low (1mg/kg Dry Matter (DM) or less), but liver and kidney cobalt concentrations increase dramatically when animals are fed high dietary cobalt [START_REF] Henry | Bioavailability of cobalt sources for ruminants, 1. Effects of time and dietary cobalt concentration on tissue cobalt concentration[END_REF]. In our study too metal content increased significantly initially 1hr (group 2) and 24 h (group 3) post dose in liver but was later found to be nearby control level after 7days of cessation of cobalt <1mg/kg DM for all the tissues. In the present study no differences in any organ tissue microscopic morphology was observed between treated and control animals, supporting the contention that the observed macroscopic changes were adaptive and reversible and probably not significant in organ physiological functions.

Apart from above parameters, an important report favoring the dose 12.5 mg Co/Kg BW to be viewed as a potential NOAEL (no observed adverse effect level) compound is that the LD 50 in rats for CoCl 2 .6H 2 O has ranged from 42.4 to 190mg Co/Kg BW in various studies (ATSDR, 2001;[START_REF] Southern | Eimeria acervulina injection in chicks fed cobalt in presence or absence of excess dietary methioinine[END_REF]. To conclude the above dose of cobalt is nontoxic and therefore may be considered for developing a suitable neutraceutical.

  chosen as blank. The absorption signal generated during atomization was recorded and compared with standards. The results were depicted as mg Co/kg Dry Matter (DM).

  followed by Dunnet's multiple comparison tests. One way ANOVA was used in analyzing body weight change, organ weight, food and water consumption, food efficiency, hematological values. When group means were significantly different (P<0.05), individual pair-wise comparisons were made using Dunnet's . Fisher's exact test was performed on all histopathology data. The analyses were performed using Graphpad Prism4 software.

  the study can be explained in the terms of the predictable physiological adaptive response to cobalt supplementation. Cobalt is known to increase erythropoeisis therefore RBC, Hb and Hct values in blood of cobalt fed animals were found to be increased in Group 2 & 3 which in turn increased the O 2

  4).Cobalt concentrations as estimated for this dose was well within range i.e.

  /reperfusion injury and chronic graft deterioration by a single-donor treatment with cobalt-protoporphyrin for the induction of heme oxygenase -1. Transplantation 2002; 74: 591-598. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-Inducible Factor 1 is a Basic-Helix-Loop-Helix-PAS Heterodimer Regulated by Cellular O2 Tension. PNAS. 1995; 92: 5510-5514. WHO. World health statistics annual 1995. Geneva: World Health Organization, 1996.Xi L, Tahler M, Yin C, Salloum F, Kukreja RC. Cobalt chloride induces delayed cardiac preconditioning in mice through selective activation of Hif-1 and AP-1 and iNOS signaling. Am.J. Physiol. heartCirc. Physiol. 2004; 287: 2369-75.

Figure1.

  Figure CaptionFigure1. Experimental design for toxicological analysis of the recommended dose of cobalt chloride (12.5 mg Co/Kg BW).

Table 1 . Survival, body weights, feed and water intake of rats supplemented with CoCl 2

 1

	Groups # Survival	Mean body weight (g) a		Mean	Water intake	Mean feed
						feed	(grand means) b	conversion
			Day 0	Day of sacrifice %Change	intake b Day	Day of	efficiency c
							0	sacrifice
	i)	8/8	205±4.5 230±6.5	25±4	15.6	21.5 25.5	0.18
				243±3.5(Group	38±4.5(Group
				1b)	1b)
	ii)	8/8	200±3.5 222±2.5	22±3	12.2	22.2 25.6	0.17
	iii)	8/8	202±4.5 225±4	23±2.5	12.5	20.5 26.6	0.16
	iv)	8/8	205±4	245±6*	40±5.5*	15.7*	20.8 27.1*	0.17*
	*with respect to Group 1 (b) control.

# Number of animals surviving at 7 days/number initially in group.

a Weight and weight changes are provided as mean± standard error (SEM).

b Feed and water intake is expressed as grams per rat per day; grand mean of daily cage means, thus no standard deviation for the mean of means.

c Feed conversion efficiency is expressed as g weight gain/ g food consumed; grand mean of daily cage means, thus no standard deviation for the mean of means.

Table 2 : Hematological analysis

 2

	Parameters	Group 1	Group 2	Group 3	Group 4
		(control)	(1hr post dose)	(24hr post dose)	(7d post dose)
	WBC	4.5± 1.1	4.56± 1.5	5.85± 1.3*	4.9± 1.1
	Lymphocytes 96.5± 2	94.45± 3	95.3± 2	98.9± 0.8
	Granulocytes 0.95± 0.6	2.7± 1.2*	1.25± 0.7	1.2± 0.13
	Monocytes	2.9± 1.5	5.5± 1.3*	3.42± 1.4	3.1± 0.1
	RBC	4.32± 0.3	6.2± 0.3*	6.74± 2.2*	6.9± 0.2*
	MCV	57.7± 2.26	58.9± 1.1	58.5± 2.4	57.4± 0.9
	MCHC	37.8± 0.7	38.1± 1.1	36.9± 2	37.3± 1
	Hct	36.4± 0.8	38.8± 1*	39.9± 1.6*	40± 1*
	Hb	13.75± 0.51 15.3± 0.6*	15.7± 0.3*	14.9± 0.6*
	PLT	37.2±1.2	37.4±2.0	40.4±1.5	39.6±1.7
	WBC=white blood cells (million mm -3 ); RBC=red blood cells (million mm -3 ); lymphocytes, granulocytes,
	monocytes expressed in %; MCV= mean cell volume and mean corpuscular hemoglobin concentration
	(MCHC) expressed in % volume; Hct= hematocrit (%); Hb= hemoglobin (g%); PLT= Platelets (lakhs mm -
	3 ) p<0.05 (*).

Table 3 : Organ-body weight index

 3

	Organ	Group 1	Group 2	Group 3	Group 4
		(control)	(1hr post dose)	(24hr post dose)	(7d post dose)
	Brain	0.77±0.04 0.79±0.06	0.81±0.06	0.81±0.05
	Heart	0.26±0.01 0.31+0.02	0.33±0.04*	0.28±0.08
	Liver	2.15±0.1	3.1±0.2*	2.83±0.1*	2.39±0.14
	Lung	0.46±0.03 0.59±0.05*	0.55±0.02*	0.48±0.05
	Kidney 0.3±0.01	0.32±0.05	0.31±0.06	0.32±0.07
	Spleen 0.16±0.01 0.17±0.03	0.17±0.08	0.18±0.02
	*p<0.05

Table 4 : Cobalt concentration (DM mg/Kg tissue) in vital organs and plasma

 4

	Cobalt (DM) Group 1	Group 2	Group 3	Group 4
		(control)	(1hr post dose)	(24hr post dose)	(7d post dose)
	Brain	0.27±0.02 0.28±0.03	0.26±0.05	0.26±0.03
	Heart	0.46±0.04 0.39+0.01	0.43±0.03	0.40±0.06
	Liver	0.71±0.05 0.85±0.02*	0.86±0.01*	0.72±0.01
	Lung	0.76±0.03 0.79±0.05	0.79±0.02	0.72±0.05
	Kidney	0.36±0.05 0.39±0.03	0.31±0.06	0.32±0.08
	Plasma	0.19±0.05 0.22±0.03	0.23±0.07	0.18±0.08
	DM= Dry matter, p<0.05 (*)

Table 5 : Biochemical analysis

 5 CK= creatine kinase, LDH= lactate dehydrogenase, SGPT= glutamic-pyruvic transaminase, SGOT= serum glutamic-oxaloacetic transaminase. *p<0.05.

	Parameters	Group 1	Group 2	Group 3		Group 4
		(control)	(1hr post dose)	(24hr	post	(7d post dose)
				dose)
	CK (UI -1 )	52.6± 2.3	58.9± 3.4	60.5± 2.9		59.3± 2.7
	Creatinine(mg dl -1 )	0.68± 0.06	0.72± 0.07	0.71± 0.03		0.67± 0.01
	Uric acid(mg dl -1 )	4.9± 1.1	5.1± 1.7	5.4± 1.3		5.3± 2
	LDH (UI -1 )	132.03±10.2	136.5± 12.2	139.3± 9.1		134.9± 7.9
	SGPT (UI -1 )	14.35± 2.3	15.4± 2.5	14.6± 2.4		14.6± 1.9
	SGOT (UI -1 )	54.7± 1.5	51.5± 5.7	55.4± 3.6		53.6± 4.2
	Glucose (g dl -1 )	48.2± 4.7	59.6± 3.4*	66.4± 7.2*		51.1± 4.7

Table 6 : Biochemical analysis

 6

	Parameters	Group 1	Group 2	Group 3	Group 4
		(control)	(1hr post dose)	(24hr post dose)	(7d post dose)
	MDA (nmol/mg protein) 0.54± 0.03 0.56± 0.03	0.52± 0.05	0.57± 0.04
	Protein (µg/µl)	2.2± 0.02	2.38± 0.04	2.4± 0.02	2.2± 0.03
	MDA= Malonaldehyde (Lipid peroxidation), *P<0.05

Acknowledgements: Ms. Kalpana is thankful to Council of Scientific and Industrial Research, New Delhi for providing the fellowship during her tenure at DIPAS and Mr. Dhananjay and Dr Kadirvelu for their assistance. The study was supported by Defence Research and Development Organization, Ministry of Defense, Govt of India.


U Kalkuhl

Deschl

N Ozaki

K A Matheis

M Gamber

T Feidl

T Nolte

A Kalkuhl
email: arno.kalkuhl@boehringer-ingelheim.com

U Deschl

Identification of genes involved in gentamicin induced nephrotoxicity in rats-a toxicogenomic investigation

Keywords: Toxicogenomics, Gentamicin, Nephrotoxicity, Kidney, Rat, Microarray, Kim-1, ATP6V1D, GATM, transferrin receptor

Identification of genes involved in gentamicin induced nephrotoxicity in rats-a toxicogenomic investigation

analysis is needed to understand associations between gene expression alterations and described pathological processes. In the current study we have chosen gentamicin as nephrotoxic model compound. The pathomechanism of this compound was studied in detail in many studies but only a limited number of transcriptional gene expression changes after gentamicin administration has been published [START_REF] Amin | Identification of putative gene based markers of renal toxicity[END_REF][START_REF] Kramer | Overview on the application of transcription profiling using selected nephrotoxicants for toxicology assessment[END_REF][START_REF] Nagy | Early gene expression in the organ of Corti exposed to gentamicin[END_REF].

Gentamicin was chosen as a nephrotoxic model compound as this compound was the object of many mechanistic investigations. Its tissue specific toxicity is considered to be caused by a selective accumulation of this drug in the tubular epithelial cells in renal cortex, where its concentration is several times higher than in the plasma [START_REF] Nagai | Molecular aspects of renal handling of aminoglycosides and strategies for preventing the nephrotoxicity[END_REF][START_REF] Goodman | Gilman's the pharmacological basis of therapeutics[END_REF]Mingeot-Leclerq and Tulkens, 1999). On the cellular level an intense binding of the drug to the brush border of the proximal tubule could be demonstrated [START_REF] Moestrup | Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs[END_REF]. After binding, gentamicin is taken into the cell by endocytosis where it inhibits the activities of lysosomal phospholipases and sphingomyelinase [START_REF] Laurent | Mechanism of aminoglycoside-induced lysosomal phospholipidosis: in vitro and in vivo studies with gentamicin and amikacin[END_REF].

Therefore, gentamicin also inhibits the degradation of phospholipid-rich cell membranes which leads to an accumulation of phospholipids. This phenomenon is reflected ultrastructurally by the appearance of myeloid or lamellar bodies implicating the occurrence of lysosomal enzyme leakage. This may explain the tubular necrosis seen after administration of gentamicin [START_REF] Bennett | Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity[END_REF]. Within one week a replicative rate increase in the proximal tubule is observed. In addition, apoptosis plays an important role in gentamicin toxicity which could also contribute to clear off damaged cells and to control the compensatory proliferative response [START_REF] Servais | Gentamicin causes apoptosis at low concentrations in renal LLC-PK1 cells subjected to electroporation[END_REF]. Finally, there is evidence for oxidative stress in gentamicin-induced nephrotoxicity.

In the current study a dose of 80 mg/kg gentamicin was given daily to male rats for one week.

Selected parameters of clinical pathology were measured and the kidney were evaluated by histopathology. Affymetrix GeneChip® microarrays were used for gene expression analysis of the kidneys.

A c c e p t e d m a n u s c r i p t MATERIALS AND METHODS

Chemicals

The study performed at Boehringer Ingelheim used gentamicin obtained from Sigma (Steinheim, Germany).

Animal Treatment and sample collection

The gentamicin study was performed by Boehringer Ingelheim Pharma GmbH & Co. KG.

Male Han Wistar rats (CrlGlxBrlHan:WI) were purchased from Charles River Laboratories (Sulzfeld, Germany). At administration start the rats were about 12 week old and weighed approximately 200 g. The animals were dosed intramuscularly in the right hind limb once daily with 80 mg/kg gentamicin (solved in 0.9% saline) for 7 days. Corresponding control rats were dosed with equivalent volumes of vehicle (0.9% saline). The animals were assigned to groups (3 rats/group) by weight using weight stratification-based computer software. The animals received a food ration in pellet form (Kliba No. 3438.0.25,Provimi Kliba SA (Kaiseraugst,Switzerland)) ad libitum but it was withdrawn in the afternoon before necropsy.

Municipal tap water (Stadtwerke Biberach; Germany) was available ad libitum via drinking bottles. The animals were kept under controlled temperature (20 ± 2°C), humidity (45-75%), and lighting (12 h light/dark cycle) and were acclimatized for a minimum of 11 days. The animals were kept in groups of 3 per cage (Noryl, type V). At necropsy the kidneys were removed and hemi-dissected. One half of the kidney were preserved in 10% buffered formaldehyde for histological examinations, the other half were frozen in liquid nitrogen for RNA isolation.

Clinical chemistry

For clinical chemistry 550µl blood per animal were collected at necropsy. Plasma creatinine and Blood Urea Nitrogen (BUN) were determined with a Hitachi 917 (405657) and Roche Diagnostics Kits (Roche Diagnostics GmbH, Mannheim).

Histopathology

Representative sections of the kidneys were processed and embedded in paraffin, sectioned at a thickness of approximately 4 µm and stained with hematoxylin-eosin (H&E), Periodic Acid Schiff reagent, and immunohistochemically for the Proliferating Cell Nuclear Antigen (PCNA) by application of the streptavidin-biotin method and diaminobenzidine as substrate.

RNA Isolation

Total RNA was isolated individually from harvested snap frozen hemi-dissected left and right kidneys using QIAGEN RNeasy kits (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions. The RNA concentration was determined spectrophotometrically (BioPhotometer, Eppendorf, Wesseling-Berzdorf). The integrity of the RNA was checked by gel analysis using RNA 6000 Nano chips on an Agilent 2100 Bioanalyzer from Agilent Technologies (Waldbronn, Germany).

Hybridization of cRNA to Oligonucleotide Arrays

Biotin-labeled cRNA samples for hybridization on Affymetrix GeneChip® RG_U34A arrays

Gene Expression Profiles

Each animal was analyzed individually on a Affymetrix RG_U34A Gene Chip. This chip contains 8740 probe sets corresponding to about 7000 annotated rat genes and 1740 expressed sequence tags (ESTs). For comparison between the gentamcin-treated group and the vehicletreated group, the Wilcoxon test was applied using the software Expressionist Analyst (Version 5.1.2, Genedata AG, 4016 Basel, Switzerland). To identify the genes that had a significant increase or decrease in intensity values a significance level of α = 0.05 was chosen.

Due to the fact that a two sided Wilcoxon rank test was used, the p-value cutoff was set at p = 0.099 which correspond to a p-value cutoff at 0.05 for a one sided Wilcoxon rank test.

Median fold changes in intensity values across the statistically significant genes were required to be ≥ 1.7-fold or ≤ -1.7-fold. This approach using a combination of a p-value cut-off with a Consortium [START_REF] Shi | The MicroArray Quality Control (MAQC) project shows inter-and intraplatform reproducibility of gene expression measurements[END_REF]. The probe sets were annotated through the use of Ingenuity

Pathways Analysis (Ingenuity Systems, Redwood City, CA).

A c c e p t e d m a n u s c r i p t

9

RESULTS

Clinical Chemistry

The clinical chemistry results of the study displayed a statistically significant increase of creatinine in blood plasma after gentamicin treatment with 80 mg/kg for 7 days. BUN levels were not affected (Table 1).

A c c e p t e d m a n u s c r i p t 10

Histopathology

Gentamicin-induced effects on the kidneys were generally minimal to moderate in severity and were degenerative, regenerative, and inflammatory in nature. Degenerative changes were most prominent in proximal convoluted tubules and consisted of vacuolization, cytoplasmic granularity and cell death by necrosis and apoptosis with cell desquamation (Figure 1). The PAS-negative cytoplasmic granularity was considered to represent the histological correlate of exaggerated lysosomal phospholipids storage. The epithelium of convoluted tubules was slightly hypercellular with increased basophilia, indicating regenerative proliferation. Hyaline tubular casts occurred occasionally. The interstitial tissue adjacent to prominently affected tubules was minimally to slightly infiltrated by lymphocytes. Affected tubules were unevenly distributed indicating that some, but not all nephrons were affected.

Correlating to the regenerative proliferation identified in H&E stained slides, PCNA immunostaining revealed more labeled nuclei in proximal convoluted tubules of animals treated at 80 mg/kg for one week when compared to controls. An increased labeling was also evident in distal convoluted tubules (Figure 1).

Gene Expression Analysis

Based on chosen selection criteria n=313 regulated probe sets were identified to be deregulated by gentamicin. Among these probe sets, 211 genes were assigned to their molecular functions, pathways and biological processes. The functions are outlined in Table 2 and are described in more detail in the following discussion part.

A c c e p t e d m a n u s c r i p t

DISCUSSION

Vesicles, endocytotic pathways and lysosomes

Gentamicin is described to be internalized by proximal tubule cells via receptor-mediated endocytosis. In our study we identified five statistically significantly deregulated genes which encode for proteins which are involved in intracellular vesicle movement (Myo1G, ARF6, RSC1A1 , EXOC3, PAFAH1B1). The observed transcriptional changes of these genes are therefore considered to reflect disturbances in vesicle movement caused by the gentamicin.

One strongly up-regulated gene of this group was Myo1G (Myosin IG), which is involved in multiple types of vesicle motility [START_REF] Allan | Motoring around the Golgi[END_REF]. ARF6 (ADP-ribosylation factor 6), which regulates clathrin-mediated endocytosis [START_REF] Wennerberg | The Ras superfamily at a glance[END_REF] and endosome movement [START_REF] Taunton | Actin filament nucleation by endosomes, lysosomes and secretory vesicles[END_REF] was also deregulated. It was described to be localized to specialized apical endosomes in kidney proximal tubules [START_REF] Maranda | Intra-endosomal pH-sensitive recruitment of the Arfnucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes[END_REF]. Further deregulated genes of this "vesicle"-group were RSC1A1 (regulatory solute carrier protein, family 1, member 1, also known as PLEKHM2), which is involved in the transport of vesicles and in maintaining the structure and position of the Golgi apparatus [START_REF] Boucrot | The intracellular fate of Salmonella depends on the recruitment of kinesin[END_REF][START_REF] Goldstein | The road less traveled: emerging principles of kinesin motor utilization[END_REF][START_REF] Clarke | Intermediate filaments: vimentin moves in[END_REF][START_REF] Allan | Motoring around the Golgi[END_REF], EXOC3 (exocyst complex component 3), which is part of the exocyst complex that mediates targeting and tethering of post-Golgi secretory vesicles [START_REF] Hsu | The exocyst complex in polarized exocytosis[END_REF] and PAFAH1B1 (platelet-activating factor acetylhydrolase, isoform Ib/ alpha subunit 45kDa), which plays a role in the retrograde movement of cytoplasmic dynein (Smith et al., 2000). This is of interest as gentamicin is transported retrograde through the secretory pathway to the Golgi complex and to the endoplasmatic reticulum (Sandoval and Molitoris, 2003). Deregulation of the megalin receptor which is described to play an important role in receptor mediated endocytosis of gentamicin [START_REF] Sandoval | Gentamicin traffics retrograde through the secretory pathway and is released in the cytosol via the endoplasmic reticulum[END_REF][START_REF] Moestrup | Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs[END_REF] were not represented on the RG_U34A Gene Chip and could not be analyzed.

A c c e p t e d m a n u s c r i p t 12

The mRNA of five genes encoding proteins which are localised in the lysosomal and endosomal compartment was found to be deregulated, among them the TFRC (transferrin receptor) gene. As TFRC is a marker for early endosomes [START_REF] Kurz | Autophagy, ageing and apoptosis: the role of oxidative stress and lysosomal iron[END_REF] the observed upregulation is considered to reflect a gentamicin effect on endocytosis. The change of TFRC mRNA might also indicate a gentamicin effect on intracellular iron-homeostasis. A protective effect of iron chelators on gentamicin toxicity was previously reported [START_REF] Walker | Evidence suggesting a role for hydroxyl radical in gentamicin-induced acute renal failure in rats[END_REF]Piruska and Schacht, 1995;[START_REF] Song | Variable efficacy of radical scavengers and iron chelators to attenuate gentamicin ototoxicity in guinea pig in vivo[END_REF]Shah and Schacht, 1996).

Interestingly, gentamicin chelates mitochondrial iron in hair cells of the inner ear, forming a very oxidant Fe(II)-gentamicin complex capable of causing cell death [START_REF] Song | Protection from gentamicin ototoxicity by iron chelators in guinea pig in vivo[END_REF].

Effects of gentamicin on lysosomal function were indicated by up-regulation of CTSL2

(cathepsin L2) and CTSD (cathepsin D), which encode lysosomal cysteine proteinases [START_REF] Ishidoh | Gene regulation and extracellular functions of procathepsin L[END_REF]Kominami, 1998, Zaidi et al., 2008). The enzymatic acitivity of Cathepsin L enzyme was previously reported to be inhibited in S1 and S2 proximal tubule cells after gentamicin administration to rats [START_REF] Olbricht | Alterations in lysosomal enzymes of the proximal tubule in gentamicin nephrotoxicity[END_REF]. Therefore the up-regulation of CTSL2 may indicate a counter-regulatory effect on mRNA level. Furthermore, the membrane transporter SLC9A3 (solute carrier family 9 (sodium/hydrogen exchanger), member 3, also known as NHE-3) was strongly downregulated on mRNA level by gentamicin. This antiporter is present in renal endosomes and plays a role in pH regulation, Na+ homeostasis and endosomal trafficking (D´Souza et al., 1998). This gentamicin effect was previously described by [START_REF] Sassen | Dysregulation of renal sodium transporters in gentamicintreated rats[END_REF] on protein level in the renal cortex of gentamicin treated rats (80mg/kg). Furthermore, we observed an up-regulation of ATP6V1D (ATPase, H+ transporting, lysosomal 34kDa, V1 subunit D), a subunit of a key enzyme for early endosomal fusion and acidification of intracellular organelles [START_REF] Hammond | Membrane potential mediates H(+)-ATPase dependence of "degradative pathway" endosomal fusion[END_REF][START_REF] Sun-Wada | Lysosome and lysosome-related organelles responsible for specialized functions in higher organisms, with special emphasis on vacuolar-type proton ATPase[END_REF]). An association between the observed up-regulation of this gene and gentamicin induced lysosomal phospholipidosis is likely as the acidic pH in the lysosomes is essential for

A c c e p t e d m a n u s c r i p t

13 the degradation of phospholipids by lysosomal hydrolases. Interestingly this ATPase is colocalized in proximal tubule cells with ARF6, which was also deregulated on mRNA level in our study.

Membrane transporters, amino acid/glucose metabolism

The mRNA expression of several genes encoding transporter proteins was significantly altered after gentamicin application in our study (Table 1). The majority of these genes were downregulated, which is interpreted as a sign of decreased kidney function. Among them two amino acid transporter, SLC6A18 (solute carrier family 6, member 18) and SLC6A19 (solute carrier family 6 (neutral amino acid transporter), member 19), were down-regulated which might reflect the observed aminoaciduria after gentamicin treatment [START_REF] Suhl | Changes in renal brush-border sodium-dependent transport systems in gentamicin-treated rats[END_REF]. The observed deregulation of amino acid metabolism genes (please refer to Table 1, Amino acid metabolism), might be a secondary effect in this context.

An effect on water reabsorption was indicated by the down-regulation of AQP2 (aquaporin 2).

The AQP2 gene encodes a water channel protein expressed only in the principal cells of renal collecting duct [START_REF] Agre | The aquaporin family of water channels in kidney[END_REF] with important functions at water reabsorption. An effect on AQP2 on protein level in the tubulointerstitial structures of the rat renal cortex was previously described after gentamicin application [START_REF] Lee | Gentamicin decreases the abundance of aquaporin water channels in rat kidney[END_REF]. The observed deregulation might contribute to the observation that gentamicin reduces the urinary concentration ability of the kidney [START_REF] Miura | Effects of gentamicin on renal function in isolated perfused kidneys from male and female rats[END_REF]). An additional down-regulated transporter was SGLT-2 (= SLC5A2; solute carrier family 5, sodium/glucose cotransporter, member 2), indicating an effect of gentamicin on renal glucose reabsorption. An effect on glucose metabolism was indicated by the observed down-regulation of G6PC (glucose 6phosphatase), a key gluconeogenic enzyme, which is expressed in the proximale tubule, but not in other segments of the nephron (Marsenic, 2009).

Translation

The expression level of 9 genes involved in the regulation and initiation of translation were found to be up-regulated (please refer to Table 1). This is of interest as gentamicin exerts its pharmacological activity mainly by inhibition of protein synthesis by binding to the prokaryotic 30S ribosomal subunit to prevent the formation of an initiation complex with messenger RNA. However, at high doses the translation of eukaryotic tissues is also affected.

It is reported that gentamicin induces translational readthrough of stop codons of a messenger RNA leading to the production of nonsense peptides [START_REF] Howard | Sequence specificity of aminoglycoside-induced stop condon readthrough: potential implications for treatment of Duchenne muscular dystrophy[END_REF] and that aminoglycosides produce mistranslation in eukaryotic tissues including the kidneys [START_REF] Manuvakhova | Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system[END_REF][START_REF] Bennett | Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity[END_REF][START_REF] Buss | Gentamicin administered in vivo reduces protein synthesis in microsomes subsequently isolated from rat kidneys but not from rat brains[END_REF][START_REF] Palmer | Mistranslation in a eucaryotic organism[END_REF]. The expression changes of the 9 genes involved in translation in our study might are therefore considered to indicate a counter-regulatory mechanism induced by effects of a nephrotoxic dose of gentamicin on translation in the kidneys.

A c c e p t e d m a n u s c r i p t Proliferation

High doses of gentamicin were described to cause tubular necrosis followed by a replicative rate increase in the proximal tubule within one week, an effect which was also seen histopathologically and immunohistochemically (PCNA-staining) in our study. In line with this effect we observed a deregulation of a number of genes which play a role in the proliferation processes such as Jun (v-jun sarcoma virus 17 oncogene), JunB (jun b protooncogene) and MYC (v-myc myelocytomatosis viral oncogene) [START_REF] Karin | From JNK to pay dirt: jun kinases, their biochemistry, physiology and clinical importance[END_REF][START_REF] Shaulian | AP-1 as a regulator of cell life and death[END_REF][START_REF] Cowling | Mechanism of transcriptional activation by the Myc oncoproteins[END_REF][START_REF] Sakisaka | The roles of nectins in cell adhesions: cooperation with other cell adhesion molecules and growth factor receptors[END_REF]. In addition, NRG1

(neuregulin 1), with a function in regenerative processes, and NUDC (nuclear distribution gene C homolog), which is described to be up-regulated in proliferating cells (Zheng et al., 1999;[START_REF] Castagnino | Neu differentiation factor/heregulin induction by hepatocyte and keratinocyte growth factors[END_REF][START_REF] Gocke | The human homologue of the Aspergillus nuclear migration gene nudC is preferentially expressed in dividing cells and ciliated epithelia[END_REF] were deregulated. PCNA was not statistically significant up-regulated on mRNA level in our study but there was a clear trend to higher mRNA expression in the gentamicin treated animals.

Cell death

Cell death by apoptosis and necrosis were evident in proximal convoluted tubules of our study, in line with the well documented fact that apoptosis plays a central role in the gentamicin induced nephrotoxicity. As possible mechanisms, destabilization of the lysosomal membrane with the subsequent release of cathepsins but also effects of cytosolic gentamicin on mitochondria or the impairment of Bax (BCL2-associated X protein) protosomal degradation were suggested [START_REF] Chwieralski | Cathepsin-regulated apoptosis[END_REF][START_REF] Servais | Renal cell apoptosis induced by nephrotoxic drugs: cellular and molecular mechanisms and potential approaches to modulation[END_REF]. In our study we found an up-regulation of CTSL2 (Cathepsin L2) which might reflect the activation of apoptosis. The observed up-regulation of Bax mRNA could be interpreted as a transcriptional response to the described impairment of the Bax proteasomal degradation. In addition, we observed a deregulation of TP53 (tumor protein p53) and several downstream targets of TP53 protein homolog (mouse)), indicating that this apoptotic pathway is also affected by gentamicin. As proliferation pathways are closely linked to apoptotic pathways a clear separation of "proliferation-genes" and "apoptosis-genes" were not possible and, therefore, these genes were listed together in Table 1.

Oxidative stress/ DNA repair mechanisms/ Stress response

Reactive oxygen species (ROS) are also discussed to participate in gentamicin-induced apoptosis [START_REF] Lesniak | Ternary complexes of gentamicin with iron and lipid catalyze formation of reactive oxygen species[END_REF]. In our study we observed the deregulation of a number of genes associated with the protection against oxidative injury. HMOX1 (heme oxygenase 1) which was unregulated was of specific interest as it was previously reported to be induced in kidney following toxic and ischemic insults [START_REF] Nath | Heme oxygenase-1: a provenance for cytoprotective pathways in the kidney and other tissues[END_REF][START_REF] Takahashi | Heme oxygenase-1: a novel therapeutic target in oxidative tissue injuries[END_REF]. APEX1

(APEX nuclease 1), a multifunctional DNA repair enzyme, and MGMT (O-6-methylguanine-DNA methyltransferase), which repairs alkylated guanine in DNA, were up-regulated in our study. In addition, we observed the up-regulation of several genes, which are involved in stress response pathways, like the transcription factor DDIT3 (DNA-damage-inducible transcript 3), several heatshock proteins (HSPA5, HSP90AA1, HSP90B1, HSPB1) and CHKA (Choline kinase alpha) [START_REF] Welch | Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease[END_REF][START_REF] Wang | Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153)[END_REF][START_REF] Chen | Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery[END_REF][START_REF] Aoyama | Structure and function of choline kinase isoforms in mammalian cells[END_REF]van de Water et al. 2006). Taken together these gene expression changes are judged to reflect a rather unspecific stress response.

Nephrotoxicity

Gentamicin induced in our study the expected tubulopathy with particular affection of proximal convoluted tubules. This was associated with a strong deregulation of several mRNAs encoding for kidney proteins which are considered to be nephrotoxicity markers. The glycoprotein serves as an earlier urinary diagnostic marker for proximal tubule injury [START_REF] Zhou | Comparison of kidney injury molecule-1 and other nephrotoxicity biomarkers in urine and kidney following acute exposure to gentamicin, mercury, and chromium[END_REF][START_REF] Han | Kidney Injury Molecule-1 (KIM-1): a novel biomarker for human renal proximal tubule injury[END_REF], Vaidya et al., 2006).In addition SPP1 (Osteopontin), ATF3

(activating transcription factor 3), Lipocalin (LCN2) and TIMP1 (TIMP metallopeptidase inhibitor 1) [START_REF] Hai | ATF3 and stress responses[END_REF][START_REF] Thukral | Prediction of nephrotoxicant action and identification of candidate toxicity-related biomarkers[END_REF][START_REF] Yuen | Ischemic and nephrotoxic acute renal failure are distinguished by their broad transcriptomic responses[END_REF][START_REF] Sieber | Comparative analysis of novel non-invasive renal biomarkers and metabonomic changes in a rat model of gentamicin nephrotoxicity[END_REF] which were described to be affected after kidney injury were found to be strongly up-regulated on mRNA level in our study. The finding of the EGF (epidermal growth factor) down-regulation was in line with the report of [START_REF] Verstrepen | Altered growth factor expression during toxic proximal tubular necrosis and regeneration[END_REF] and was of interest as EGF downregulation is frequently observed after acute renal failure [START_REF] Perco | Protein biomarkers associated with acute renal failure and chronic kidney disease[END_REF].

We also observed a strong deregulation of several genes that were previously associated with nephrotoxicity of gentamicin. The down-regulation of KLK1 (Kallikrein 1) after gentamicin administration was previously described by [START_REF] Amin | Identification of putative gene based markers of renal toxicity[END_REF]. In addition gentamicin-treated rats were shown to have strongly reduced levels of urinary Kallikrein [START_REF] Higa | Role of the prostaglandin and kallikrein-kinin systems in aminoglycoside-induced acute renal failure[END_REF]. In addition, a renal protective effect of Kallikrein against gentamicin-induced nephrotoxicity was reported [START_REF] Murakami | Human kallikrein gene delivery protects against gentamycininduced nephrotoxicity in rats[END_REF][START_REF] Bledsoe | Role of Tissue Kallikrein in Prevention and Recovery of Gentamicin-induced Renal Injury[END_REF]. The observed changes of ICAM1

(intercellular adhesion molecule 1 (CD54)) might also be associated with nephrotoxicity. The deregulation of this gene was previously observed in gentamicin treated rats via RT-PCR [START_REF] Bledsoe | Role of Tissue Kallikrein in Prevention and Recovery of Gentamicin-induced Renal Injury[END_REF]) and was also reported to be up-regulated after cisplatin induced acute renal failure and in experimental tubulointerstitial nephritis [START_REF] Tang | Cytokine expression, upregulation of intercellular adhesion molecule-1, and leukocyte infiltration in experimental tubulointerstitial nephritis[END_REF][START_REF] Kelly | Protection from toxicant-mediated renal injury in the rat with anti-CD54 antibody[END_REF][START_REF] Perco | Protein biomarkers associated with acute renal failure and chronic kidney disease[END_REF]Safirstein, 2004).

Furthermore we observed a reduced mRNA level of GATM (L-arginine:glycine amidinotransferase). A connection to nephrotoxicity is suggested by the fact that a decreased expression of GATM was also detected in the kidney of cisplatin treated rats [START_REF] Amin | Identification of putative gene based markers of renal toxicity[END_REF]. GATM catalyzes the transfer of a guanido group from arginine to glycine, forming

A c c e p t e d m a n u s c r i p t

18 guanidinoacetic acid, the immediate precursor of creatine, which is the rate-limiting step of creatine biosynthesis in kidney [START_REF] Wyss | Creatine and creatinine metabolism[END_REF]. Interestingly, a decrease in urinary excretion of guanidinoacetic acid was reported as a sensitive marker of gentamicin nephrotoxicity in rats (Kiyatake et al., 2006;[START_REF] Kiyatake | Urinary guanidinoacetic acid excretion as an indicator of gentamicin nephrotoxicity in rats[END_REF][START_REF] Takeda | Guanidinoacetic acid (GAA) synthesis in rat tubular suspension as a system for evaluating gentamicin (GM) nephrotoxicity[END_REF] A possible interrelation between the reduced GATM mRNA level and the observed increased plasma concentration of creatinine, which was measured as known marker for renal function in our study, must be discussed as well. There is increasing evidence that increased plasma creatinine levels are associated with increased plasma creatine levels and it was shown that the serum concentration of creatine is increased in many patients with cronic renal failure (Jones and Burnet 1974;[START_REF] Mitch | Creatinine metabolism in chronic renal failure[END_REF][START_REF] Wyss | Creatine and creatinine metabolism[END_REF]. In rats increased plasma creatine levels resulted in a decrease of GATM mRNA, protein expression and enzymatic activity. This effect was described as a pretranslational feedback repression [START_REF] Guthmiller | Cloning and sequencing of rat kidney Larginine:glycine amidinotransferase. Studies on the mechanism of regulation by growth hormone and creatine[END_REF][START_REF] Van Pilsum | The antagonistic action of creatine and growth hormone on the expression of the gene for rat kidney L-arginine:glycine amidinotransferase[END_REF]. Therefore the observed down-regulation of GATM after treatment with gentamicin on mRNA expression level might be explained by a feedback repression of GATM by creatine. In conclusion this suggests that GATM and the above discussed reaction-product guanidinoacetic acid are interesting candidate markers for nephrotoxicity.

GSTM1 (glutathione S-transferase M1) gene encodes GST Yb1 protein which was described as urinary leakage biomarker for distal tubule damage [START_REF] Kilty | Histologically defined biomarkers in toxicology[END_REF]. Interestingly the GSTM1 mRNA level was down-regulated after gentamicin treatment in this study. Downregulation of GSTM1 mRNA was also described in a study comparing gene expression patterns of GSTs in kidney and liver of iron-overloaded mice [START_REF] Souza | Immunohistological analysis of glutathione transferase A4 distribution in several human tissues using a specific polyclonal antibody[END_REF] which indicates a relation between GSTM1 mRNA down-regulation and general oxidative stress mechanisms in kidney.

In contrast CALB1 (calbindin 1, 28kDa), considered to detect specifically distal tubular or

A c c e p t e d m a n u s c r i p t

19 collecting duct damage [START_REF] Steiner | Cyclosporine A decreases the protein level of the calciumbinding protein calbindin-D 28kDa in rat kidney[END_REF][START_REF] Takashi | Urinary 28-kD calbindin-D as a new marker for damage to distal renal tubules caused by cisplatin-based chemotherapy[END_REF] was not deregulated in our study. Albumin, Cystatin C or ß-microglobulin which are used as urinary markers for nephrotoxicity due to increased glomerular filtration or decreased reabsorption were not expected and not found to be deregulated in the kidney after gentamicin administration.

It is worth discussing that a mRNA based analysis with microarrays has some limitations.

Several potential events could arise between the mRNA and the final protein, e.g.

translational regulation and differences in protein in vivo half-lives [START_REF] Greenbaum | Comparing protein abundance and mRNA expression levels on a genomic scale[END_REF][START_REF] Beyer | Post-transcriptional expression regulation in the yeast Saccharomyces cerevisiae on a genomic scale[END_REF]. Furthermore changes on phosphorylation cascades as well as posttranslational modifications and metabolites can not be detected. Nevertheless up to now microarray analysis is the tool of choice for mechanistic analysis as the number of annotated molecules is very high when compared to other techniques. With genomics it is possible to gain an overall picture of intracellular processes. The high number of measured endpoints has the advantage that hypothesis are not generated based on only one or a minimal number of gene or protein deregulation. Rather it is possible to strengthen the value of the gained information by verifying a hypothesis with a big group of analyzed molecules.

In summary, we could identify 211 genes which showed significant changes at the mRNA level in response to a nephrotoxic dose of gentamicin. The majority of the described genes are correlated for the first time with gentamicin toxicity. Based on the mechanistic knowledge of gentamicin toxicity from the literature, it was possible to hypothesize a potential damage sequence in accordance with histopathological observations. Deregulation of lysosomal genes or genes involved in vesicle trafficking could reflect accumulation of gentamicin in lysosomes of proximal tubule epithelial cells which might be a primary effect of gentamicin. Several of the genes involved here were previously reported to be expressed in proximal tubule cells (e.g. CTSL2, ARF6 and ATP6V1D). An acute stress on proximal tubule cells was also

  it possible to study genome-wide expression profiles in response to the administration of pharmaceutical compounds and xenobiotics. The description of altered gene expression offers the possibility to understand the mode of action and to gain new insight into side effects at the molecular level. There is also the potential to study complex mechanisms of toxicity within a single animal study. Bridging between previous knowledge generated by classical biochemical or pathological work and gene expression

  organelles like lysosomes and endosomes. The overload of phospholipid membranes may result in compromised lysosomal membrane integrity which might lead to

  to the GeneChip Expression Analysis Technical Manual from Affymetrix Inc. (Santa Clara, CA). Briefly, 5 µg total RNA was used for cDNA synthesis with the SuperScript Choice System from Invitrogen Life Technologies, (Invitrogen GmbH, Karlsruhe, Germany) employing a modified T7 primer with 24 thymidines at the 5' end. After spin column purification, biotin-labeled cRNA was synthesized from the cDNA using the ENZO RNA Transcript Labeling Kit (Affymetrix Inc., Santa Clara, CA). Spin columnpurified cRNA was quality controlled using an Agilent 2100 Bioanalyzer and spectrophotometrically quantified. The cRNA (15 µg) was then fragmented in buffer supplied with the GeneChip Sample Cleanup Module and hybridized for 16 h at 45°C. The microarrays were washed and stained with streptavidin-phycoerythrin (Dianova, Hamburg, Germany) on the Affymetrix Fluidics Station 400. Fluorescent images were read using the Affymetrix GeneChip Scanner. The raw data image files (DAT) were converted into CEL files using the Affymetrix Microarray Suite 5.0.

  -off was chosen based on the results of the Micro Array Quality Control

  Several genes with functions in fatty acid metabolism , like ACAA1 (acetyl-Coenzyme A acyltransferase 1), ACADM (acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain), ACADSB (acyl-Coenzyme A dehydrogenase, short/branched chain) and ACSL3 (acyl-CoA synthetase long-chain family member 3) were found to be down-regulated ("Fatty acid metabolism", Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com). The observed down-regulation of these genes is considered to reflect a disturbance in lipid degradation by gentamicin. Whether these changes in lipid-metabolism are connected with the gentamicin-related inhibition of phospholipid-degradation or are due to other toxic effects of this compound on the kidney cells remains open.

  g. CCNG1 (cyclin G1), BTG2 (BTG family, member 2), MDM2 (Mdm2 p53 binding

  with an up-regulation of 38-fold after gentamicin administration was the Kim-1 (Kidney injury molecule-1, also known as HAVCR1) gene. This type I cell membrane

  responses and likely real damage responses further downstream of the damage cascade, i.e. by deregulation of genes associated with cell death together with proliferation genes. Which effects like the deregulation of nephrotoxicity marker genes as well as fatty acid metabolism genes are primary, secondary or even tertiary responses to direct gentamicin influence would need a time response investigation. Furthermore, additional studies are needed to validate these associations and to use toxicogenomics for safety evaluation of new compounds. An example for this type of analysis is the study of[START_REF] Wang | Validation of putative genomic biomarkers of nephrotoxicity in rats[END_REF], where a subset of genes was investigated after administration of several nephrotoxins including gentamicin.

Table 1 Clinical chemistry results for plasma creatinine and blood urea nitrogen (BUN) levels

 1

		Dose [0mg/kg]		Dose [80mg/kg]		Δ%
	Animal	BUN	Creatinine		BUN	Creatinine
	Nr.	[mmol/L]	[µmol/L]	Animal Nr.	[mmol/L]	[µmol/L]	BUN	Creatinine
	111	9.29	59.2 311	10.99	72.7
	113	9.61	57.4 312	10.22	73.7
	114	10.08	60.5 313	8.9	68.8
	Mean	9.66	59.03 Mean	10.04	71.73*	3.90%	21.51%
	* statistical significant (p< 0.01)

Table 2 Classification of significant gene expression changes in the rat kidney induced by gentamicin (80 mg/kg/day). Platform: Affymetrix RG_U34A. Deregulated genes with an expression change ≥ 1.7-fold and ≤ -1.7 respectively and a p<0.01 (Wilcoxon rank test) are shown.

 2

	Gene Symbol	Entrez Gene Name	Affymetrix ID	FC
	Amino acid metabolism
	ABAT	4-aminobutyrate aminotransferase	D87839_g_at	-1.70
	ALDH1A1	aldehyde dehydrogenase 1 family, member A1	AF001898_at	-1.74
	ASL	argininosuccinate lyase	D13978_s_at	2.10
	ASL	argininosuccinate lyase	AB016536_s_at	1.81
	ASNSD1	asparagine synthetase domain containing 1	rc_AA799531_at	1.75
	CBS	cystathionine-beta-synthase	M88347_s_at	-1.79
	CDO1	cysteine dioxygenase, type I	E03229cds_s_at	-2.23
	DDC	dopa decarboxylase (aromatic L-amino acid decarboxylase)	M84648mRNA_s_at	-2.63
	GNMT	glycine N-methyltransferase	rc_AA893219_at	-1.84
	GNMT	glycine N-methyltransferase	X06150cds_at	-1.72
	GNMT	glycine N-methyltransferase	X06150cds_g_at	-1.99
	HSD17B4	hydroxysteroid (17-beta) dehydrogenase 4	rc_AI101743_s_at	-1.79
	KMO	kynurenine 3-monooxygenase (kynurenine 3-hydroxylase)	AF056031_at	-2.30
	KYNU	kynureninase (L-kynurenine hydrolase)	U68168_at	-2.34
	MPST	mercaptopyruvate sulfurtransferase	D50564_at	-1.78
	NEDD4	neural precursor cell expressed, developmentally down-regulated 4	U50842_at	1.73
	PHGDH	phosphoglycerate dehydrogenase	X97772_g_at	-1.79
	PSAT1	phosphoserine aminotransferase 1	rc_AI102868_at	-2.45
	PSAT1	phosphoserine aminotransferase 1	rc_AI102868_g_at	-2.79
	Cell death/ Proliferation
	AGTR1	angiotensin II receptor, type 1	M86912exon_g_at	-2.34
	AGTR1	angiotensin II receptor, type 1	M74054_s_at	-2.26
	AGTR1	angiotensin II receptor, type 1	M86912exon_at	-2.38
	BAX	BCL2-associated X protein	S76511_s_at	1.86
	BAX	BCL2-associated X protein	U59184_at	1.83
	BTG2	BTG family, member 2	rc_AA944156_s_at	3.96
	BTG2	BTG family, member 2	M60921_at	2.08
	BTG2	BTG family, member 2	M60921_g_at	5.24
	BTG3	BTG family, member 3	AF087037_g_at	2.66
	BTG3	BTG family, member 3	AF087037_at	1.97
	CCNG1	cyclin G1	X70871_at	5.09
	CDC2L5	cell division cycle 2-like 5 (cholinesterase-related cell division controller)	rc_AA875127_g_at	2.00
	CDC2L5	cell division cycle 2-like 5 (cholinesterase-related cell division controller)	rc_AA875127_at	1.92


Juan Francisco Sánchez Muñoz-Torrero

Paloma Barquilla

Raul Velasco

Maria Del Carmen

Fernández Capitan

MD. Lucia Nazaret Pacheco

Lucia Vicente

Jose Luis Chicón

MD Sara Jose Trejo

Jose Zamorano

Alicia Lorenzo Hernandez

Sánchez Muñoz-Torrero

Juan Francisco

H San

Internal Medicine Barquilla Pedro Alcántara

Internal Medicine Fernandez Pedro Alcantara

Maria Capitan

H La Del Carmen

Internal Medicine Paz

Nararet ; H Pacheco

Pedro San

Internal Medicine Vicente Lucia Alcantara

Jose Luis

Medicine Zamorano

Pedro De Alcantara

Lorenzo Hernandez

H La Paz

Juan Fco

PhD Md

MD.. Raul Barquilla Paloma

MD Velasco Carmen Fernández

PhD Capitan * Md

Ph.D Alicia Zamorano

Lorenzo Hernandez

MD, Ph D Juan Fco Sánchez Muñoz-Torrero

Adverse drug reactions in internal medicine units and associated risk factors

Keywords: Adverse drug reactions, incidence, risk factors. Word count (abstract) : 243 Word count article (excluding abstract, references, tables) : 2454

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche

 [START_REF] Rawlins | Mechanisms of adverse drug reactions[END_REF]was used to establish the potential for predicting suspected adverse reactions. The algorithm of Naranjo [START_REF] Naranjo | A method for estimating the probability of adverse drug reactions[END_REF] and co-workers was used to establish the causality between the drug and the suspected adverse reaction. ADR was considered if it was definitive or probable.Data were collected using a specific data collection sheet developed for this investigation. For all patients, information regarding age, sex, weight and tall, duration of hospital stay, all diagnosis at discharge was collected, as well as amounts and brand names of medicines taken in the previous month, during the hospitalization, including self -medication. Blood test for creatinina, ALT, AST, alkaline fosfatase and filtration glomerular rate (estimated by Cockroft-Gault formula:GFR(ml/min)=(140-age(y)) x body weight(kg)/72 x serum creatinine (mg/dl); in female x 0.85) ) were determinate at entry and the day of detection of ADRs. Patients were then classified into (Group 1) patients without ADR, (Group 2) patients admitted because of ADR or detected during hospitalization.

The possible interactions between drugs were evaluated using Lexi-Interact™ Online (Lexi-Comp, Inc. Ohio) [START_REF]Lexi-Comp. Drug interactions handbook, and drug interactions software[END_REF] . This is a comprehensive Drug-to-Drug Interaction Analysis Program with a Rapid indicator to show the interaction data. This program also assigned a risk rating of A, B, C, D, or X. The progression from A to X is accompanied by increased urgency for responding to the data. In general, A and B monographs are of academic, but not clinical concern.

Monographs rated C, D, or X always requires the user's attention. interact with each other in a clinically significant manner. The benefits of concomitant use of these two medications usually outweigh the risks. An appropriate monitoring plan should be implemented to identify potential negative effects. Dosage adjustments of one or both agents may be needed in a minority of patients. Monitor Therapy. D: Data demonstrate that the two medications may interact with each other in a clinically significant manner. A patient-specific assessment must be conducted to determine whether the benefits of concomitant therapy outweight the risks. Specific actions must be taken in order to realize the benefits and/or minimize the toxicity resulting from concomitant use of the agents. These actions may include aggressive monitoring, empiric dosage changes, choosing alternative agents. Consider Therapy Modification. X: Data demonstrate that the specified agents may interact with each other in a clinically significant manner. The risks associated with concomitant use of these agents usually outweigh the benefits. These agents are generally considered contraindicated. Avoid

Combination. The severity of ADRS was defined as: Minor (effects would be considered tolerable; in most cases no need for medical intervention); Moderate (medical intervention needed to treat effects; effects do not meet criteria for Major); and Major (effects may result in death, hospitalization, permanent injury, or therapeutic failure). We also collected about the need of prolong hospitalization and the drug interaction itself as the cause of hospitalization.

Data analysis. Data analysis. Data analysis. Data analysis. All the data were expressed as the mean±SD or percentage. The differences in the values of the variables between the start and the end of the 10 -weeks treatment period were calculated as percent changes, values at the end of 12 week's treatment minus those at the start of the treatment x 100/the value at the start of the treatment. Patients who were hospitalized because of ADRS were excluded for the study of potential factors associated with ADRS. The Chi-square test and Student's t-test for independent samples were used to assess the statistical significance between ratios and means, respectively. In order to control potential confounding factors and to determine independent association between ADRS and risk factors for ADRS, the logistic regression analysis was employed. Factors demonstrating significant association with an ADR were included in the multivariable logistic regression model. Since other researchers had identified sex and age as risk factors for an ADR, these variables were included in the model without considering the statistical significance of the univariate analysis. A p value lower to 0.05 was considered statistically significant. . . . Data analysis was performed using the SPSS software package (SPSS Inc., Chicago, Illinois, USA).

Results:

Results: Results: Results: During a 10-weeks study period in two university hospitals, 405 patients were intensively monitored with respect to ADRs. (Fig. 1). There were 128 adverse reactions in 126 (31%) patients. In 24 patients the ADRs was the cause of hospitalization and 102 patients suffered ADRs during the hospitalization in internal medicine ward. The ADRs were moderate in 93 (73%), major in 22 (17%) and were fatal in 2 patients (1, 6%). ADRs in our study.

The frequency of ADRs in hospitals varies greatly between different published studies [START_REF] Pirmohamed | Adverse drug reaction[END_REF][START_REF] Davies | Adverse drug reactions in hospitals: a narrative review[END_REF][START_REF] Trontell | Expecting the unexpected: drug safety, pharmacovigilance, and the prepared mind[END_REF] , which can be explained by differences in their methods of identifying ADRs, the definition of adverse reactions, the methodology used for their detection, the complexity level of the pathologies under treatment and the severity of the reaction, among others [START_REF] Talley | Drug-induced illness[END_REF][START_REF] Klein | The frequency of adverse drug reactions as dependent upon age, sex and duration of hospitalization[END_REF][START_REF] Martin | et als Age and sex distribution of suspected adverse drug reactions to newly marketed drugs in general practice in England: analysis of 48 cohort studies[END_REF][START_REF] Krähenbühl-Melcher | Drug-related problems in hospitals: a review of the recent literature[END_REF][START_REF] Schein | Epidemiology, outcomes research, and drug interactions Drug[END_REF][START_REF] Hazell | Under-reporting of adverse drug reactions : a systematic review[END_REF][START_REF] Lazarou | A Meta-analysis of Prospective Studies Incidence of Adverse Drug Reactions in Hospitalized[END_REF][START_REF] Brvar | The frequency of adverse drug reaction related admissions according to method of detection, admission urgency and medical department specialty[END_REF] . The rate of ADRs found in this study -31%-was higher than the average of 6.7% (range 1.2-24%) found in previous studies [START_REF] Pirmohamed | Adverse drug reaction[END_REF][START_REF] Davies | Adverse drug reactions in hospitals: a narrative review[END_REF][START_REF] Trontell | Expecting the unexpected: drug safety, pharmacovigilance, and the prepared mind[END_REF] but consistent with other reports [START_REF] Zopf | Risk factors associated with adverse drug reactions following hospital admission: a prospective analysis of 907 patients in two German university hospitals[END_REF][START_REF] Camargo | Adverse drug reactions: a cohort study in internal medicine units at a university hospital[END_REF] using, like us, the "gold standard of pharmacovigilance", intensive prospective ADRs detection [START_REF] Zopf | Risk factors associated with adverse drug reactions following hospital admission: a prospective analysis of 907 patients in two German university hospitals[END_REF][START_REF] Camargo | Adverse drug reactions: a cohort study in internal medicine units at a university hospital[END_REF] . Drugs causing ADRs [START_REF] Bond | Adverse Drug Reactions in United States Hospitals[END_REF] were similar to those described by other authors [START_REF]Lexi-Comp. Drug interactions handbook, and drug interactions software[END_REF][START_REF] Camargo | Adverse drug reactions: a cohort study in internal medicine units at a university hospital[END_REF][START_REF] Bond | Adverse Drug Reactions in United States Hospitals[END_REF] where cardiovascular drugs, anticoagulants and antibiotics were most frequently associated with ADRs. Reactions affecting the gastrointestinal tract, skin, mucosa and haematological system are among the most frequently observed events. The ADRs are in relation with drugs used for managing frequently disease of patients admitted in Internal Medicine wards.

Previous studies have examined many potential risk factors associated with ADRs in a variety of settings [START_REF] Talley | Drug-induced illness[END_REF][START_REF] Mcdonnell | Hospital admissions resulting from preventable adverse drug reactions[END_REF][START_REF] Zopf | Gender-based differences in drug prescription: relation to adverse drug reactions[END_REF] . Conditions often cited as potential risk factors of ADRs are increasing age, sex, large numbers of prescribed drugs in individual patients or length of hospitalisation [START_REF] Talley | Drug-induced illness[END_REF][START_REF] Lagnaoui | Adverse drug reactions in a department of systemic diseases-oriented internal medicine: prevalence, incidence, direct costs and avoidability[END_REF][START_REF] Mcdonnell | Hospital admissions resulting from preventable adverse drug reactions[END_REF][START_REF] Klein | The frequency of adverse drug reactions as dependent upon age, sex and duration of hospitalization[END_REF][START_REF] Martin | et als Age and sex distribution of suspected adverse drug reactions to newly marketed drugs in general practice in England: analysis of 48 cohort studies[END_REF][START_REF] Zopf | Gender-based differences in drug prescription: relation to adverse drug reactions[END_REF] . Failure of dose adjustment in patients with impaired renal function and, drug-drug interactions also were as important predictors of ADRs [START_REF] Zopf | Gender-based differences in drug prescription: relation to adverse drug reactions[END_REF][START_REF] Krähenbühl-Melcher | Drug-related problems in hospitals: a review of the recent literature[END_REF][START_REF] Schein | Epidemiology, outcomes research, and drug interactions Drug[END_REF] . Studies in the field of pharmacovigilance using multiple regression models are rare and, we unknown what variables were independent ADR predictors [START_REF] Zopf | Risk factors associated with adverse drug reactions following hospital admission: a prospective analysis of 907 patients in two German university hospitals[END_REF] . There is consensus about the predictive power of large numbers of prescribed drugs -more than four-as an independent predictor of ADRs [START_REF] Lagnaoui | Adverse drug reactions in a department of systemic diseases-oriented internal medicine: prevalence, incidence, direct costs and avoidability[END_REF][START_REF] Klein | The frequency of adverse drug reactions as dependent upon age, sex and duration of hospitalization[END_REF][START_REF] Zopf | Risk factors associated with adverse drug reactions following hospital admission: a prospective analysis of 907 patients in two German university hospitals[END_REF] . In our analysis, number of drug interactions were one of the most important factors associated with apparition of ADRs, but not the number of drugs itself. We found a high rate of medication use with interaction by cytocrome P450 2C9,2D6 and 3A4. These cytocrome frequently are implicate in metabolism of common drugs [START_REF] Flockhart | Drug Interactions and Cytochrome P450 System: The role of Cytochrome P450 2C19[END_REF] . Nowadays, complexity of treatments make difficult to know the possibility of apparition of interactions between two or more drugs, so adequate software package [START_REF] Flockhart | Drug Interactions and Cytochrome P450 System: The role of Cytochrome P450 2C19[END_REF][START_REF] Szekendi | Active surveillance using electronic triggers to detect adverse events in hospitalized patients[END_REF][START_REF] Bobb | The epidemiology of prescribing errors: the potential impact of computerized prescriber order entry[END_REF] will be useful in this way. Most studies summarized that 69% of adverse events as preventable [START_REF] Mcdonnell | Hospital admissions resulting from preventable adverse drug reactions[END_REF][START_REF] Bond | Adverse Drug Reactions in United States Hospitals[END_REF] and can therefore be considered to result from medication errors. Although they are several recommendations about using electronic devices, their use have not been generalized and electronic devices have several problems that have to be removed [START_REF] Flockhart | Drug Interactions and Cytochrome P450 System: The role of Cytochrome P450 2C19[END_REF][START_REF] Bobb | The epidemiology of prescribing errors: the potential impact of computerized prescriber order entry[END_REF] . We suggest checking drug interactions on a software package, or assess the cytocrome involve in metabolism of drugs most frequently .

Other common problem that has not been previously well studied is renal function as cause of ADRS [START_REF] Bobb | The epidemiology of prescribing errors: the potential impact of computerized prescriber order entry[END_REF][START_REF] Lundkvist | Pharmacoeconomics of adverse drug reactions[END_REF] [START_REF] Bobb | The epidemiology of prescribing errors: the potential impact of computerized prescriber order entry[END_REF] .Dose adjustment with renal function probably would have prevented the apparition of several ADRs. Although, we can observe ADRs before identify the changes in renal function, prevent dose titration. Sex, age and malnutrition were also correlated with the presence of ADRs in literature reports [START_REF] Talley | Drug-induced illness[END_REF][START_REF] Lagnaoui | Adverse drug reactions in a department of systemic diseases-oriented internal medicine: prevalence, incidence, direct costs and avoidability[END_REF][START_REF] Mcdonnell | Hospital admissions resulting from preventable adverse drug reactions[END_REF][START_REF] Klein | The frequency of adverse drug reactions as dependent upon age, sex and duration of hospitalization[END_REF][START_REF] Martin | et als Age and sex distribution of suspected adverse drug reactions to newly marketed drugs in general practice in England: analysis of 48 cohort studies[END_REF][START_REF] Zopf | Gender-based differences in drug prescription: relation to adverse drug reactions[END_REF] . However, in our population, neither albumin, sex or increasing age were independent ADR predictors. In summary, acute changes in renal function or drug interactions were observed in 88% of patients suffering ADRs. Therefore we suggest that drug doses adjustment for renal function and assess interactions could be prevenible a great number of ADRs in clinical practice.

In terms of pharmacoeconomy it was calculated that cost of hospitalization of patient with

ADRs could be about two thousand of million dollars per year in the United States [START_REF] Lundkvist | Pharmacoeconomics of adverse drug reactions[END_REF] . It is estimated that patients who had an adverse drug reaction, the duration of hospitalization was prolonged by 4,3 days compared with patients who did not experience an adverse drug reaction leading to an increase in costs for each episode of hospitalization. In the present study, lenght of hospitalization was greater for ADRS (18 vs 9,6 days). We can´t analyze if a longer duration was the cause or the consequence of ADRs. However, the fact that ADRs appeared in the 5 day of hospitalization in most cases, shows that ADRs can be one of the main causes of a longer duration of being hospitalized for ADRs patients.

The study have some limitations. Probably the most important is the fact that we couldn´t analyzed specifically the factor implicated in ADRs in each particular case. Despite using a multiple regression model, a definitive discrimination between causative or confounding factors in predicting an ADR is difficulty. We can not judge if renal failure was cause or effect of ADRs in all situations. Also, we only analysed pharmacokinetic and no pharmacodynamic interactions due of program used. However, renal insufficiency or drug interaction were implicated in nine of

4 PATIENTS

 4 AND METHODS. PATIENTS AND METHODS. PATIENTS AND METHODS. PATIENTS AND METHODS. This prospective observational study included all patients admitted in two General Internal Medicine wards of 2 Docents Hospitals , San Pedro de Alcantara (Cáceres) and La Paz (Madrid) in Spain, over a period of 10 consecutive weeks from 1 September 2009 to 14 November 2009. For detection and evaluation of potential ADRS all admissions underwent daily intensive pharmacovigilance by at minimum three physicians (1 staff physician and 2 residents in training). Classification of adverse drug reactions Classification of adverse drug reactions Classification of adverse drug reactions Classification of adverse drug reactions. The classification proposed by Rawlins and Thompson

Table 1

 1

	Among patients who had ADRs during admission, (were excluded from analysis those
	hospitalized for this reason), factors associated with the aparition of ADRs were analyzed and
	showed in table 3. Renal function and drug-drug interaction were statiscally significative (p<
	0.001). Elevated serum creatinine at hospitalization was significantly higher in patients at ADRs

shows main characteristics of patients in the study. There were no significant differences between the group of ADRs and non ADRs in age, sex and BMI. Number of diagnoses of each patient was bigger in patients with ADRs: 2, 3±1.2 vs. 2.0±1.1. ADRs patients have more lung diseases (35% vs. 25%, p< 0.05) and diabetes than controls (35% vs. 20%, p < 0.001). An important difference was the duration of hospitalization that was longer for ADRs patients: 18±17 days vs. 9.6±5.8, p<0.001. Time from hospitalization to ADRs was 4.3±3.9 days.

Table

2

shows the drugs causing the different ADRs. 28 patients suffered from diarrhea because of antibiotic use. The second most frequent ADRs was bleeding, because of acenocumarol or enoxaparin (15 patients). The third frequent ADRs was rash (9 patients), although cutaneous involvement was the ADRs in 20 patients, mainly due to some antibiotics but also fenitoin and atorvastatin. than more high in control group during hospitalization (mg/dL): 2.02±1.22 vs1.72 ± 0.95. A filtration glomerular rate below of 60 ml/min was observed in 41% of patients with ADRs vs 22%

In the logistic regression analysis to analyze factors associated with ADRs we found (as shown in table 4): the duration of hospitalization (p< 0.001), change of 20% of filtration glomerular rate (p< 0.001) and any drug interaction were associated with ADR (p< 0.0001).

The most common ADRs in hospitalized patients and factors associated were analyzed in this study. Diarrhea postantibiotic, bleeding due to antithrombotic therapy and rash were the most frequent ADRs observed. Conditions associated with ADRs during hospitalization were prolonged hospitalization, acute change in filtration glomerular rate and drug interaction. Other previously cited as risk factors, like age, sex and number of drugs used were not related with

  . We report an elevated incidence of renal dysfunction measured as having

	glomerular filtration rate below 60 ml/min, 41% in ADRS patients vs 22% controls. Also we
	observe a worsening of renal function during hospitalization, renal function drop >20% in 35% of
	case vs 18% controls. This important prevalence of renal impairment was clearly different from
	reported by others
	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 1 :

 1 10 ten ADRs, so we have to consider these two mechanisms as very important factors contributing to ADRs development. Studies with a longer period of observation and more hospitals implicated in the study of ADRs would help to determine the significance of these mechanisms in ADRs or identifying more factors implicated. Baseline Characteristics of Patients.

	In summary, we believe that the present study makes important contributions. First, the real
	incidence of ADRs in Internal Medicine could have been infraestimated. Second, acute renal
	function worsening during hospitalization, prior renal dysfunction and drug interactions seem to
	r o F be important factors contributing to ADRs appearance.
	P
	e
	e r
	R
	e
	v i e
	w

Table 3 :

 3 Differences in Patients with and without an Adverse Drug Reaction observed during hospitalization. Estimated glomerular filtration rate *: Grade >C denote interaction between drugs from a clinically significant manner to agents considered contraindicated (for more details see Methods)

	CONTROL	CASE	p value
	N=279	N=102

Table 4 :

 4 Logistic regression analysis with ADRs observed during hospitalization as dependent variable. Estimated glomerular filtration rate *: Grade >C denote interaction between drugs from a clinically significant manner to agents considered contraindicated (for more details see Methods)

	Odds ratio	95% CI	p value

EGFR:

European Journal of Clinical Pharmacology