Effects of the anxiolytic benzodiazepine oxazepam on freshwater gastropod reproduction: a prospective study.

Psychoactive drugs have emerged as contaminants over the last few decades. These drugs are frequently prescribed and poorly eliminated by wastewater treatment plants, and many are present at non-negligible concentrations in surface waters. Several studies have investigated the non-target organism toxicity of one such drug, oxazepam, a benzodiazepine anxiolytic frequently detected in rivers. However, very little is known about the impact of this drug on reproduction. We investigated the effects of environmentally relevant concentrations of oxazepam on Radix balthica, a freshwater gastropod widespread in Europe. We identified the reproductive organs of Radix balthica. We then exposed this gastropod to oxazepam for two months and assessed several reproductive parameters, from reproductive organ status to behavioral parameters. We found that adults exposed to 10 µg/L oxazepam display an increase in the density of spermatozoa, and that adults exposed to 0.8 µg/L oxazepam displayed a decrease in the number of eggs per egg mass over time. By contrast, oxazepam had no effect on shell length, the size of male reproductive organs or social interactions. Finally, a locomotor activity analysis showed the distance covered over time decreased in all conditions of exposure to oxazepam, potentially reflecting a disturbance of exploratory activity. These results shed light on the effects of oxazepam on the reproduction of a non-target freshwater mollusk.

Low concentrations of oxazepam induce feeding and molecular changes in Radix balthica juveniles.

Psychotropics, especially benzodiazepines, are commonly prescribed worldwide. Poorly eliminated at wastewater treatment plants, they belong to a group of emerging contaminants. Due to their interaction with the GABAA receptor, they may affect the function of the nervous system of non-target organisms, such as aquatic organisms. The toxicity of oxazepam, a very frequently detected benzodiazepine in continental freshwater, has been largely studied in aquatic vertebrates over the last decade. However, its effects on freshwater non-vertebrates have received much less attention. We aimed to evaluate the long-term effects of oxazepam on the juvenile stage of a freshwater gastropod widespread in Europe, Radix balthica. Juveniles were exposed for a month to environmentally-relevant concentrations of oxazepam found in rivers (0.8 µg/L) and effluents (10 µg/L). Three main physiological functions were studied: feeding, growth, and locomotion. Additionally, gene expression analysis was performed to provide insights into toxicity mechanisms. There was a strong short-term activation of the feeding rate at low concentration, whereas the high dose resulted in long-term inhibition of food intake. A significant decrease in mortality rate was observed in juveniles exposed to the lowest dose. Shell growth and locomotor activity did not appear to be affected by oxazepam. Transcriptomic analysis revealed global over-expression of genes involved in the nervous regulation of the feeding, digestive, and locomotion systems after oxazepam exposure. The molecular analysis also revealed a possible interference of animal manipulation with the molecular effects induced by oxazepam exposure. Overall, these results improve our understanding of the effects of the psychoactive drug oxazepam on an aquatic mollusc gastropod.

Bioconcentration and behavioral effects of four benzodiazepines and their environmentally relevant mixture in wild fish.

We studied the adverse effects of four benzodiazepines frequently measured in European surface waters. We evaluated bioaccumulation potential of oxazepam, bromazepam, temazepam, and clobazam in freshwater fish species - perch (Perca fluviatilis) and we conducted a series of behavioral trials to assess their potential to alter boldness, activity, and social behavior. All selected endpoints were studied individually for each target benzodiazepine and as a mixture of all tested compounds to assess possible combinatory effects. We used a three-dimensional automated tracking system to quantify the fish behavior. The four compounds bioconcentrated differently in fish muscle (temazepam > clobazam > oxazepam > bromazepam) at high exposure (9.1, 6.9, 5.7, 8.1 µg L-1, respectively) and low exposure (0.5, 0.5, 0.3, 0.4 µg L-1, respectively) concentrations. A significant amount of oxazepam was also measured in fish exposed to temazepam, most likely because of the metabolic transformation of temazepam within the fish. Bromazepam, temazepam, and clobazam significantly affected fish behavior at high concentration, while no statistically significant changes were registered for oxazepam. The studied benzodiazepines affected behavior in combination, because the mixture treatment significantly changed several important behavioral traits even at low concentration, while no single compound exposure had such an effect at that dose. Based on our results, we conclude that effects of pharmaceuticals on aquatic environments could be underestimated if risk assessments only rely on the evaluation of single compounds. More studies focused on the combinatory effects of environmentally relevant mixtures of pharmaceuticals are necessary to fill the gaps in this knowledge.

Embryotoxicity of ozonated diclofenac, carbamazepine, and oxazepam in zebrafish (Danio rerio).

Pharmaceutical residues are polluting the surface water environments worldwide. Sewage and wastewater treatment, therefore, needs to be improved in order to remove pharmaceutical residues from the effluent. One such treatment improvement is effluent ozonation. Even though ozonation has proven to be very efficient in reducing pharmaceutical parent compound concentrations in wastewater effluents, much remains unclear regarding potentially toxic ozonation by-product (OBP) formation. In this study, we sought to elucidate the aquatic toxicity of ozonated pharmaceuticals in zebrafish (Danio rerio) embryos in a static 144 h post fertilization (hpf) fish embryotoxicity (ZFET) assay. Three pharmaceuticals commonly detected in wastewater effluents, i.e. carbamazepine, diclofenac, and oxazepam, were selected for testing. Toxicity was assessed before and after 1 min ozonation (0.053 mg L-1 peak O3 concentration) and 10 min ozonation (0.147 mg L-1 peak O3 concentration). Chemical analysis showed that carbamazepine and diclofenac were largely removed by ozone (90 ±â€¯11% and 97 ±â€¯3.8%), whereas oxazepam was removed to a lesser extent (19 ±â€¯5.7%). The ZFET assay revealed diverging toxicities. Diclofenac embryotoxicity decreased with increasing ozonation. Oxazepam did not cause embryotoxicity in the ZFET assay either pre- or post ozonation, but larvae swimming activity was affected at 144 hpf. Carbamazepine embryotoxicity, on the other hand, increased with increasing ozonation. Chemical analysis showed the formation of two OBPs (carbamazepine-10,11-epoxide and 10,11-dihydrocarbamazepine), possibly explaining the increased embryotoxicity. The results of this study highlight the importance of new chemical and toxicological knowledge regarding the formation of OBPs in post-ozonated effluents.

No evidence of increased growth or mortality in fish exposed to oxazepam in semi-natural ecosystems.

An increasing number of short-term laboratory studies on fish reports behavioral effects from exposure to aquatic contaminants or raised carbon dioxide levels affecting the GABAA receptor. However, how such GABAergic behavioral modifications (GBMs) impact populations in more complex natural systems is not known. In this study, we induced GBMs in European perch (Perca fluviatilis) via exposure to a GABA agonist (oxazepam) and followed the effects on growth and survival over one summer (70days) in replicated pond ecosystems. We hypothesized that anticipated GBMs, expressed as anti-anxiety like behaviors (higher activity and boldness levels), that increase feeding rates in laboratory assays, would; i) increase growth and ii) increase mortality from predation. To test our hypotheses, 480 PIT tagged perch of known individual weights, and 12 predators (northern pike, Esox lucius) were evenly distributed in 12 ponds; six control (no oxazepam) and six spiked (15.5±4µgl-1 oxazepam [mean±1S.E.]) ponds. Contrary to our hypotheses, even though perch grew on average 16% more when exposed to oxazepam, we found no significant difference between exposed and control fish in growth (exposed: 3.9±1.2g, control: 2.9±1g [mean±1S.E.], respectively) or mortality (exposed: 26.5±1.8individuals pond-1, control: 24.5±2.6individuals pond-1, respectively). In addition, we show that reduced prey capture efficiency in exposed pike may explain the lack of significant differences in predation. Hence, our results suggest that GBMs, which in laboratory studies impact fish behavior, and subsequently also feeding rates, do not seem to generate strong effects on growth and predation-risk in more complex and resource limited natural environments.

Environmental relevant levels of a benzodiazepine (oxazepam) alters important behavioral traits in a common planktivorous fish, (Rutilus rutilus).

Environmental pollution by pharmaceuticals is increasingly recognized as a major threat to aquatic ecosystems worldwide. A complex mix of pharmaceuticals enters waterways via treated wastewater effluent and many remain biochemically active after the drugs reach aquatic systems. However, to date little is known regarding the ecological effects that might arise following pharmaceutical contamination of aquatic environments. One group of particular concern is behaviorally modifying pharmaceuticals as seemingly minor changes in behavior may initiate marked ecological consequences. The aim of this study was to examine the influence of a benzodiazepine anxiolytic drug (oxazepam) on key behavioral traits in wild roach (Rutilus rutilus) at concentrations similar to those encountered in effluent surface waters. Roach exposed to water with high concentrations of oxazepam (280 µg/L) exhibited increased boldness, while roach at low treatment (0.84 µg/L) became bolder and more active compared to control fish. Our results reinforce the notion that anxiolytic drugs may be affecting fish behavior in natural systems, emphasizing the need for further research on ecological impacts of pharmaceuticals in aquatic systems and development of new tools to incorporate ecologically relevant behavioral endpoints into ecotoxicological risk assessment.

An innovative and integrative assay for toxicity testing using individual fish embryos. Application to oxazepam.

This paper describes the development of an integrative embryo-toxicity assay in Japanese medaka allowing analysis of several toxicological endpoints together in a same individual. In this assay, embryos are topically exposed, and survival, hatching success, malformations, biometry, behaviour, and target gene expression are subsequently analysed in each individual. This assay was applied to oxazepam, an anxiolytic pharmaceutical compound currently found in wastewater treatment plant effluent. Even if oxazepam accumulation in embryos was very low, it caused spinal and cardiac malformations, delayed growth, erratic swimming and deregulation of genes involved in apoptosis, DNA repair and mitochondrial metabolism. Relationship between gene deregulation, abnormal behaviour, and developmental anomalies was demonstrated. This assay is sensitive enough to detect adverse effects at low chemical concentrations and at multiple endpoints in a unique fish embryo. This integrative embryo-toxicity assay is a powerful tool to characterize the spectrum of effects of new chemicals and also to link effects induced at different molecular, tissue and physiological levels.

De novo transcriptome sequencing and analysis of freshwater snail (Radix balthica) to discover genes and pathways affected by exposure to oxazepam.

Pharmaceuticals are increasingly found in aquatic ecosystems due to the non-efficiency of waste water treatment plants. Therefore, aquatic organisms are frequently exposed to a broad diversity of pharmaceuticals. Freshwater snail Radix balthica has been chosen as model to study the effects of oxazepam (psychotropic drug) on developmental stages ranging from trochophore to hatching. In order to provide a global insight of these effects, a transcriptome deep sequencing has been performed on exposed embryos. Eighteen libraries were sequenced, six libraries for three conditions: control, exposed to the lowest oxazepam concentration with a phenotypic effect (delayed hatching) (TA) and exposed to oxazepam concentration found in freshwater (TB). A total of 39,759,772 filtered raw reads were assembled into 56,435 contigs having a mean length of 1579.68 bp and mean depth of 378.96 reads. 44.91% of the contigs have at least one annotation. The differential expression analysis between the control condition and the two exposure conditions revealed 146 contigs differentially expressed of which 144 for TA and two for TB. 34.0% were annotated with biological function. There were four mainly impacted processes: two cellular signalling systems (Notch and JNK) and two biosynthesis pathways (Polyamine and Catecholamine pathways). This work reports a large-scale analysis of differentially transcribed genes of R. balthica exposed to oxazepam during egg development until hatching. In addition, these results enriched the de novo database of potential ecotoxicological models.

Home alone-The effects of isolation on uptake of a pharmaceutical contaminant in a social fish.

A wide range of biologically active pharmaceutical residues is present in aquatic systems worldwide. As uptake potential and the risk of effects in aquatic wildlife are directly coupled, the aim of this study was to investigate the relationships between stress by isolation, uptake and effects of the psychiatric pharmaceutical oxazepam in fish. To do this, we measured cortisol levels, behavioral stress responses, and oxazepam uptake under different stress and social conditions, in juvenile perch (Perca fluviatilis) that were either exposed (1.03µgl-1) or not exposed to oxazepam. We found single exposed individuals to take up more oxazepam than individuals exposed in groups, likely as a result of stress caused by isolation. Furthermore, the bioconcentration factor (BCF) was significantly negatively correlated with fish weight in both social treatments. We found no effect of oxazepam exposure on body cortisol concentration or behavioral stress response. Most laboratory experiments, including standardized bioconcentration assays, are designed to minimize stress for the test organisms, however wild animals experience stress naturally. Hence, differences in stress levels between laboratory and natural environments can be one of the reasons why predictions from artificial laboratory experiments largely underestimate uptake of oxazepam, and other pharmaceuticals, in the wild.

Anti-anxiety drugs and fish behavior: Establishing the link between internal concentrations of oxazepam and behavioral effects.

Psychoactive drugs are frequently detected in the aquatic environment. The evolutionary conservation of the molecular targets of these drugs in fish suggests that they may elicit mode of action-mediated effects in fish as they do in humans, and the key open question is at what exposure concentrations these effects might occur. In the present study, the authors investigated the uptake and tissue distribution of the benzodiazepine oxazepam in the fathead minnow (Pimephales promelas) after 28 d of waterborne exposure to 0.8 µg L-1 , 4.7 µg L-1 , and 30.6 µg L-1 . Successively, they explored the relationship between the internal concentrations of oxazepam and the effects on fish exploratory behavior quantified by performing 2 types of behavioral tests, the novel tank diving test and the shelter-seeking test. The highest internal concentrations of oxazepam were found in brain, followed by plasma and liver, whereas muscle presented the lowest values. Average concentrations measured in the plasma of fish from the 3 exposure groups were, respectively, 8.7 ± 5.7 µg L-1 , 30.3 ± 16.1 µg L-1 , and 98.8 ± 72.9 µg L-1 . Significant correlations between plasma and tissue concentrations of oxazepam were found in all 3 groups. Exposure of fish to 30.6 µg L-1 in water produced plasma concentrations within or just below the human therapeutic plasma concentration (HT PC) range in many individuals. Statistically significant behavioral effects in the novel tank diving test were observed in fish exposed to 4.7 µg L-1 . In this group, plasma concentrations of oxazepam were approximately one-third of the lowest HT PC value. No significant effects were observed in fish exposed to the lowest and highest concentrations. The significance of these results is discussed in the context of the species-specific behavior of fathead minnow and existing knowledge of oxazepam pharmacology. Environ Toxicol Chem 2016;35:2782-2790. © 2016 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Effect of bioconcentration and trophic transfer on realized exposure to oxazepam in 2 predators, the dragonfly larvae (Aeshna grandis) and the Eurasian perch (Perca fluviatilis).

Psychoactive substances are used worldwide and constitute one of the most common groups of pharmaceutical contaminants in surface waters. Although these pharmaceuticals are designed to be efficiently eliminated from the human body, very little is known about their trophic-transfer potential in aquatic wildlife. Therefore, the goal of the present study was to quantify and compare uptake of an anxiolytic (oxazepam) from water (bioconcentration) and via the consumption of contaminated diet (trophic transfer) in 2 common freshwater predators: Eurasian perch (Perca fluviatilis) and the dragonfly larvae Aeshna grandis. Bioconcentration and trophic transfer of oxazepam were found in both predator species. However, higher bioconcentrations were observed for perch (bioconcentration factor [BCF], 3.7) than for dragonfly larvae (BCF, 0.5). Perch also retained more oxazepam from consumed prey (41%) than dragonfly larvae (10%), whereas the relative contribution via prey consumption was 14% and 42% for perch and dragonflies, respectively. In addition, bioconcentration was negatively correlated with perch weight, indicating that exposure levels in natural contaminated environments differ between individuals of different size or between different developmental stages. Hence, trophic transfer of pharmaceuticals may indeed occur, and estimates of environmental exposures that do not consider intake via food or size-dependent bioconcentration may therefore lead to wrongful estimations of realized exposure levels in natural contaminated ecosystems.

Dilute concentrations of a psychiatric drug alter behavior of fish from natural populations.

Environmental pollution by pharmaceuticals is increasingly recognized as a major threat to aquatic ecosystems worldwide. A variety of pharmaceuticals enter waterways by way of treated wastewater effluents and remain biochemically active in aquatic systems. Several ecotoxicological studies have been done, but generally, little is known about the ecological effects of pharmaceuticals. Here we show that a benzodiazepine anxiolytic drug (oxazepam) alters behavior and feeding rate of wild European perch (Perca fluviatilis) at concentrations encountered in effluent-influenced surface waters. Individuals exposed to water with dilute drug concentrations (1.8 micrograms liter(-1)) exhibited increased activity, reduced sociality, and higher feeding rate. As such, our results show that anxiolytic drugs in surface waters alter animal behaviors that are known to have ecological and evolutionary consequences.

Gene expression and mutation assessment provide clues of genetic and epigenetic mechanisms in liver tumors of oxazepam-exposed mice.

Liver tumors from a previous National Toxicology Program study were examined using global gene expression and mutation analysis to define the mechanisms of carcinogenesis in mice exposed to oxazepam. Five hepatocellular adenomas and 5 hepatocellular carcinomas from male B6C3F1 mice exposed to 5000 ppm oxazepam and 6 histologically normal liver samples from control animals were examined. One of the major findings in the study was upregulation of the Wnt/ß-catenin signaling pathway. Genes that activate ß-catenin, such as Sox4, were upregulated, whereas genes that inhibit Wnt signaling, such as APC and Crebbp, were downregulated. In addition, liver tumors from oxazepam-exposed mice displayed ß-catenin mutations and increased protein expression of glutamine synthetase, a downstream target in the Wnt signaling pathway. Another important finding in this study was the altered expression of oxidative stress-related genes, specifically increased expression of cytochrome p450 genes, including Cyp1a2 and Cyp2b10, and decreased expression of genes that protect against oxidative stress, such as Sod2 and Cat. Increased oxidative stress was confirmed by measuring isoprostane expression using mass spectrometry. Furthermore, global gene expression identified altered expression of genes that are associated with epigenetic mechanisms of cancer. There was decreased expression of genes that are hypermethylated in human liver cancer, including tumor suppressors APC and Pten. Oxazepam-induced tumors also exhibited decreased expression of genes involved in DNA methylation (Crebbp, Dnmt3b) and histone modification (Sirt1). These data suggest that formation of hepatocellular adenomas and carcinomas in oxazepam-exposed mice involves alteration of the Wnt signaling pathway, oxidative stress, and potential epigenetic alterations.

Follow-up testing of rodent carcinogens not positive in the standard genotoxicity testing battery: IWGT workgroup report.

At the Plymouth Third International Workshop on Genotoxicity Testing in June 2002, a new expert group started a working process to provide guidance on a common strategy for genotoxicity testing beyond the current standard battery. The group identified amongst others "Follow-up testing of tumorigenic agents not positive in the standard genotoxicity test battery" as one subject for further consideration [L. Müller, D. Blakey, K.L. Dearfield, S. Galloway, P. Guzzie, M. Hayashi, P. Kasper, D. Kirkland, J.T. MacGregor, J.M. Parry, L. Schechtman, A. Smith, N. Tanaka, D. Tweats, H. Yamasaki, Strategy for genotoxicity testing and stratification of genotoxicity test results-report on initial activities of the IWGT Expert Group, Mutat. Res. 540 (2003) 177-181]. A workgroup devoted to this topic was formed and met on September 9-10, 2005, in San Francisco. This workgroup was devoted to the discussion of when it would be appropriate to conduct additional genetic toxicology studies, as well as what type of studies, if the initial standard battery of tests was negative, but tumor formation was observed in the rodent carcinogenicity assessment. The important role of the standard genetic toxicology testing to determine the mode of action (MOA) for carcinogenesis (genotoxic versus non-genotoxic) was discussed, but the limitations of the standard testing were also reviewed. The workgroup also acknowledged that the entire toxicological profile (e.g. structure-activity relationships, the nature of the tumor finding and metabolic profiles) of a compound needed to be taken into consideration before the conduct of any additional testing. As part of the meeting, case studies were discussed to understand the practical application of additional testing as well as to form a decision tree. Finally, suitable additional genetic toxicology assays to help determine the carcinogenic MOA or establish a weight of evidence (WOE) argument were discussed and formulated into a decision tree.

On the mechanism of hepatocarcinogenesis of benzodiazepines: evidence that diazepam and oxazepam are CYP2B inducers in rats, and both CYP2B and CYP4A inducers in mice.

The aim of this study was to evaluate diazepam and oxazepam as cytochrome P450 inducers at doses previously shown to cause liver tumors in mice but not rats. In rats, diazepam and oxazepam induced CYP2B, and were as effective as phenobarbital despite lacking phenobarbital's tumor-promoting effect in rats. In mice, diazepam and oxazepam induced both CYP2B and CYP4A at dietary doses associated with liver tumor formation. It remains to be determined why diazepam and oxazepam induce CYP4A in mice but not rats and whether this difference accounts for the apparent species difference in the tumor-promoting activity of diazepam and oxazepam.

Chemical-induced atrial thrombosis in NTP rodent studies.

Cardiac thrombosis, one of the causes of sudden death throughout the world, plays a principal role in several cardiovascular diseases, such as myocardial infarction and stroke in humans. Data from studies of induction of chemical thrombosis in rodents help to identify substances in our environment that may contribute to cardiac thrombosis. Results for more than 500 chemicals tested in rodents in 2-year bioassays have been published as Technical Reports of the National Toxicology Program (NTP) http://ntp-server.niehs.nih.gov/index. We evaluated atrial thrombosis induced by these chemical exposures and compared it to similarly induced lesions reported in the literature. Spontaneous rates of cardiac thrombosis were determined for control Fischer 344 rats and B6C3F1 mice: 0% in rats and mice in 90-day studies and, in 2-year studies, 0.7% in both genders of mice, 4% in male rats, and 1% in female rats. Incidences of atrial thrombosis were increased in high-dosed groups involving 13 compounds (incidence rate: 20-100%): 2-butoxyethanol, C.I. Direct Blue 15, bis(2-chloroethoxy)methane, diazoaminobenzene, diethanolamine, 3,3'-dimethoxybenzidine dihydrochloride, hexachloroethane, isobutene, methyleugenol, oxazepam, C.I. Pigment Red 23, C.I. Acid Red 114, and 4,4'-thiobis(6-t-butyl-m-cresol). The main localization of spontaneously occurring and chemically induced thromboses occurred in the left atrium. The literature survey suggested that chemical-induced atrial thrombosis might be closely related to myocardial injury, endothelial injury, circulatory stasis, hypercoagulability, and impaired atrial mechanical activity, such as atrial fibrillation, which could cause stasis of blood within the left atrial appendage, contributing to left atrial thrombosis. Supplementary data referenced in this paper are not printed in this issue of Toxicologic Pathology. They are available as downloadable files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 33(5), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

[Investigation of a fetal heart-rate pattern that shows a reduced oscillation amplitude]. / Silentes CTG--Hypoxie oder Intoxikation?

A fetal heart-rate pattern that has a reduced oscillation amplitude may indicate a physiological fetal dormant period but could also be an indication of fetal hypoxemia. In some rare cases such a fetal heart rate-pattern can be an indicator of cerebral or cardial fetal malformation or of an intoxication caused by sedative drugs. Our patient is a 32-year-old Para III in the phase of 29 weeks and 3 days gestation. Upon admission to the clinic, the fetal heart-rate pattern showed a reduced oscillation amplitude, and there were no signs of fetal movement. The ultrasound examination gave us no reason to suspect fetal malformation, and the results of the Doppler ultrasonography were also normal. However, although the patient had denied taking any medication at all, the results of an toxicological blood test confirmed our suspicion of benzodiazepine intoxication. Throughout the night the fetal heart-rate pattern was continuously measured, and in the early hours of the morning, after breaking down of the oxazepam medication, a normalization of the fetal heart-rate pattern was observed. This case report definitively demonstrates that Doppler ultrasonography is a valuable method for assessing any uncertainty regarding a fetal heart-rate pattern.

Changes in global gene and protein expression during early mouse liver carcinogenesis induced by non-genotoxic model carcinogens oxazepam and Wyeth-14,643.

We hypothesized that the mouse liver tumor response to non-genotoxic carcinogens would involve some common early gene and protein expression changes that could ultimately be used to predict chemical hepatocarcinogenesis. In order to identify a panel of genes to test, we analyzed global differences in gene and protein expression in livers from B6C3F1 mice following dietary treatment with two rodent carcinogens, the benzodiazepine anti-anxiety drug oxazepam (2500 p.p.m.) and the hypolipidemic agent Wyeth (Wy)-14,643 (500 p.p.m.) compared with livers from untreated mice. Male mice were exposed for 2 weeks and 1, 3 or 6 months to oxazepam or Wy-14,643 in an age-matched study design. By histopathological evaluation, no liver preneoplastic foci or tumors were detected at 6 months in treated or control groups. By cDNA microarray analysis [NIEHS Mouse Chip (8700 genes); n = 3 individual livers/group, four hybridizations/sample], expression of 36 genes or 220 genes were changed relative to control livers following 6 months of oxazepam or Wy-14,643 treatment, respectively. To obtain a more comprehensive picture of gene/protein expression changes, we also conducted a proteomics study by 2D-gel electrophoresis followed by matrix assisted laser desorption/ionization-mass spectrometry on cytoplasmic, nuclear, and microsomal subcellular fractions of the same liver samples utilized for the cDNA microarray analysis. Real-time PCR, western blot analysis and immunohistochemistry were utilized for validation and to expand the results to other time points. Cyp2b20, growth arrest- and damage-inducible gene beta (Gadd45beta), tumor necrosis factor alpha-induced protein 2 and insulin-like growth factor binding protein 1 (Igfbp5) genes and proteins were upregulated by oxazepam, and Cyp2b20, Cyclin D1, proliferating cell nuclear antigen, Igfbp5, Gadd45beta and cell death-inducing DNA fragmentation factor alpha subunit-like effector A exhibited higher expression after Wy-14,643 treatment. Most of these genes/proteins were also deregulated at 2 weeks. There appeared to be more distinct than common changes in the expression of carcinogenesis-related genes/proteins between the two compounds, suggesting that the major carcinogenic pathways are different for these compounds and may be distinct for different chemical classes.

[Genotoxicity of oxazepam--the micronucleus cytochalasin-B test]. / Genotoksicnost oxazepama--mikronukleus citohalazin-b test.

Oxazepam (OX) is one of the number benzodiazepines used therapeutically as a sedative-hypnotic and anti-anxiety agent. OX is also a common metabolite of many other benzodiazepines. Since they are among the most widely prescribed drug, the evaluation of their potential genotoxic activity should be considered very important. In the present study cytochalasin B-blocked binucleate human lymphocytes have been used to measure micronucleus induction after treatment with oxazepam. The frequency of micronuclei (MN) were analysed in 72 hours culture of peripheral blood human lymphocytes. OX was added to the cultures at the beginning of the cultivation period. The concentrations in cultures was 0.5; 5; 10; 25 and 50 mg/ml. It was found that all used oxazepam concentrations induced formation of micronuclei. MN frequencies were significantly increased (except for concentration 0.5 mg/ml OX) in relation to the control (cultures without OX). Number of binuclear lymphocytes with micronuclei, as well as number of micronuclei per a binuclear lymphocytes stand in correlation with the strength of tested concentrations--there was a dose-response. On the basis of our results we can conclude that long time administrated of OX may represent a potential hazard to human health.

Phenobarbital, oxazepam and Wyeth 14,643 cause DNA damage as measured by the Comet assay.

Although phenobarbital, oxazepam and Wyeth 14,643 are carcinogens that do not form DNA adducts, they induce mutations in the Big Blue transgenic mouse model. The mutations produced by these compounds were predominantly G-->T and G-->C transversions that we suspect arose from oxidative damage to DNA. To test this, we employed the single cell electrophoresis (Comet) assay that detects alkali-labile lesions in cells sustaining DNA damage. Human myeloid leukemia K562 cells were treated with non-cytotoxic doses of the above compounds for 3 h, then placed on slides containing low melting point agarose. Cells were lysed, exposed to alkaline buffer, electrophoresed and analyzed by microscopy for the existence of DNA damage. Extensive DNA damage, most likely due to the existence of single- and double-strand breaks and apurinic/apyrimidinic (AP) sites, was observed in cells exposed to oxazepam (1 mM) and Wyeth 14,643 (0.5 mM). On the other hand, damage of this sort was not observed in cells exposed to phenobarbital (1 mM). However, the addition of S9 liver extracts to cells exposed in the presence of phenobarbital resulted in significant amounts of DNA damage. We conclude from these studies that two of the three compounds evaluated in this study mediate their mutagenic effects through oxidative stress, but that the mechanism of DNA damage caused by phenobarbital differs from that elicited by oxazepam and Wyeth 14,643.