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.

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.

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.

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.

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.

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.

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.

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.

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.

Impaired acquisition of swimming navigation in adult mice exposed prenatally to oxazepam.

Prenatally administered oxazepam (OX) impairs adult radial maze performance in mice, possibly by permanent hippocampal changes. CDI mice were tested in swimming navigation, a sensitive indicator for hippocampal damage. Ten males and ten females were exposed to OX on fetal days 12-16 by maternal administration PO of 30 mg/kg/day and fostered at birth to untreated dams, while control mice received vehicle solution. All mice were tested at 8-9 weeks for ability to find a submerged platform in a fixed location (acquisition: 18 trials, 6 trials per day) and for capacity to re-orient towards a new platform position (reversal: 12 trials, 6 trials per day). OX mice showed a slight but significant impairment of swimming navigation during the initial part of training, as indicated by longer swimming paths during the fourth and fifth trial (day 1), an impairment due both to delayed habituation to the novel stressfull condition and acquisition of platform climbing but unrelated to navigational abilities. No treatment-dependent differences were observed in the reversal phase. During reversal, both OX and control females spent significantly more time in swimming across the location of the old platform. Unrelated to navigational performance, females showed a slightly but significantly higher swimming speed than males. Due to the absence of any navigational impairment, data suggest that prenatal exposure to oxazepam exerts long-term influence on adult learning capacities primarily through interaction with brain systems located outside the hippocampus.

Short-, medium-, and long-term effects of prenatal oxazepam on neurobehavioural development of mice.

A benzodiazepine (oxazepam) was given to nulliparous mice on days 12-16 of pregnancy, and the development and young adult behaviour of the offspring were studied. Experiment 1, using 5, 15, and 50 mg/kg doses given PO twice daily, showed a dose-dependent retardation of postnatal development of several responses such as righting, bar holding, limb placing, and auditory startle. These changes were maximal in the first 2 postnatal weeks and then were markedly attenuated, or disappeared, being apparently related to a temporary retardation of body growth. A reduction of locomotor activity at 60 days was found only in the 50 mg/kg group. The effects of the 15 mg/kg dose on postnatal body growth and neurobehavioural development were replicated in Experiments 2 and 3. Moreover, in these experiments prenatal oxazepam reduced open field activity at 14-16 days and attenuated the hyperactivity induced by dl-amphetamine sulphate (2 mg/kg IP). On the other hand activity, habituation, and response to a scopolamine challenge (2 mg/kg IP) at 21-23 days were not significantly different from those of appropriate controls. Experiment 3, using a cross-fostering procedure, showed that postnatal maternal effects were not responsible for the changes so far mentioned. Experiment 2 also investigated the acquisition of several go-no go avoidance discriminations in a shuttle-box, using either light (L) or buzzer noise (N) as the "go" signal, a compound "no go" signal (NL in the L-"go" groups and LN in the N-"go" groups), and either an extinction or a passive avoidance contingency during the "no go" signal (4 weeks of training, starting at 60 days).(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicity and carcinogenicity studies of oxazepam in the Fischer 344 rat.

Oxazepam and related benzodiazepines are used in the treatment of anxiety. Carcinogenicity studies of oxazepam were performed with the F344 rat because of marked differences in tumor responses observed in NTP studies with B6C3F1 and Swiss-Webster mice compared to the results of Sprague-Dawley rat studies submitted to the FDA by a manufacturer to support registration of the drug. Groups of 50 male and 50 female F344/N rats were fed diets containing 0, 625, 2500, or 5000 ppm oxazepam for up to 105 weeks. A stop-exposure group of 50 males and 50 females received 10,000 ppm oxazepam in diet for 26 weeks, after which animals received control diet. All 5000- and 10, 000-ppm stop-exposure males died before the end of the study. Survival of 2500-ppm males and females was lower than that of controls. Body weight gains of 2500- and 5000-ppm males and females were less than those of controls. Male rats exposed to 2500 ppm had an increased incidence of renal tubule adenoma and hyperplasia. In addition, the incidences of renal tubule adenoma and hyperplasia were increased in the 10,000-ppm stop-exposure group. The incidences of nephropathy in exposed females were greater than those in controls, and the severity of nephropathy increased in exposed males. Epithelial hyperplasia and chronic inflammation of the nonglandular stomach were increased in males given 2500 and 5000 ppm and the incidence of ulcers of the nonglandular stomach in 2500-ppm males was also greater than that in controls. In males exposed to 5000 ppm, mineralization of the glandular stomach and erosion of the duodenum were observed. In females exposed to 2500 ppm, the incidences of epithelial hyperplasia, chronic inflammation, and ulcers of the nonglandular stomach and the incidence of erosion in the glandular stomach were increased. The incidences of centrilobular hepatocyte hypertrophy in males and females given 2500 and 5000 ppm were greater than those in controls. In summary, there was equivocal evidence of carcinogenicity in males based on increased renal tubule adenomas in groups which also had significantly enhanced nephropathy. There was no evidence of carcinogenicity of oxazepam in females given a diet containing 625, 2500, or 5000 ppm for 2 years or 10,000 ppm for 6 months.

Prenatal benzodiazepine effects in mice: postnatal behavioral development, response to drug challenges, and adult discrimination learning.

Oxazepam treatment of primiparous mouse dams on days 12-16 of pregnancy (15 mg/kg p.o. twice daily) produced a transient retardation of postnatal body growth and neurobehavioral development, a reduction of the hyperactivity response to amphetamine in open-field tests on postnatal days 14-16, and a selective impairment of adult active avoidance in four go-no go discrimination tasks. Equally important for understanding the nature of the prenatal benzodiazepine syndrome were several negative results, namely, the absence of changes in homing performance on postnatal day 10, an intact hyperactivity response to scopolamine on postnatal days 21-23, a lack of effects on adult activity, and a normal passive avoidance performance in the go-no go tasks. A modification in monoaminergic regulatory functions may account for the overall profile of prenatal drug effects. Based on the results of this experiment, of a preliminary multidose study (0-50 mg/kg), and of an additional cross-fostering experiment, several methodological issues are addressed. These include the choice of appropriate treatment schedules and of testing procedures adequate for each developmental stage, and the control for various confounding variables such as litter effects, postnatal maternal influences, and developmental history.

New models for assessing carcinogenesis: an ongoing process.

Traditionally, the use of rodent models in assessing the carcinogenic potential of chemicals has been expensive and lengthy, and the relevance of the carcinogenic effect to humans is often not fully understood. Today, however, with the rapid advances in molecular biology, genetically altered mice containing genes relevant to humans (e.g. oncogenes, tumor suppressor genes) and reporter genes (e.g. lacI) provide powerful tools for examining specific chemical-gene interactions thereby allowing a better understanding of the mechanisms of carcinogenesis in a shorter period of time. This paper will cover an overview of ongoing validation efforts, followed by examples of studies using several genetically engineered models including the p53def mouse model and the Big Blue transgenic mouse model. Specifically, examples where transgenic models were integrated into the testing program based on specific hypotheses dealing with genetic alterations in cancer genes and reporter genes will be discussed. The examples will highlight possible ways genetically altered mice may be integrated into a comprehensive research and testing strategy and thereby provide an improved estimation of human health risks.

Activity of hepatic drug metabolizing enzymes following oxazepam-dosed feed treatment in B6C3F1 mice.

Oxazepam has been determined to be a potent hepatocarcinogen in mice. Evidence in the literature indicates that oxazepam is capable of inducing drug metabolizing enzymes in rodents and an association between enzyme induction and carcinogenesis has been proposed for other compounds such as phenobarbital. We examined the pattern of enzyme induction that occurs under bioassay conditions in male B6C3F1 mice. The results indicate that oxazepam is capable of inducing multiple drug metabolizing enzymes under bioassay conditions. Closer examination of the most induced samples suggests that oxazepam is a phenobarbital-type enzyme inducer.

Toxicokinetics of oxazepam in rats and mice.

The comparative toxicokinetics of oxazepam were studied in F344 rats, B6C3F1 mice, and Swiss-Webster mice of both sexes after an i.v. dose of 20 mg/kg and oral gavage doses of 50, 200, and 400 mg/kg. In addition, the toxicokinetics of oxazepam in a 3-week dosed-feed study of male B6C3F1 mice at 125 and 2500 ppm were also investigated. Results indicated that the elimination of oxazepam from plasma after i.v. injection in both rats and mice were first-order and could be best described by a two-compartment model with a terminal elimination half-life of 4-5 h for rats and 5-7 h for mice. After oral gavage dosing the peak oxazepam plasma concentrations in most rodents were reached within 2-3.5 h. At all doses studied, female rodents had significantly higher plasma concentrations than males. Absorption of oxazepam was significantly extended at higher oral doses of 200 and 400 mg/kg. At 50 mg/kg, the bioavailability of oxazepam in rats (< 50%) was lower than in Swiss-Webster mice (> 80%). The bioavailability of oxazepam in both B6C3F1 and Swiss-Webster mice decreased with increasing dose. A dose proportionality of Cmax was not observed in rats and mice after gavage doses of 50, 200, and 400 mg/kg. Plasma concentrations of oxazepam in the dosed-feed study increased with the concentration of oxazepam in the feed, a quasi-steady-state of plasma concentrations of oxazepam was reached after approximately 4 days ad libitum exposure. In B6C3F1 mice, the estimated relative bioavailability of oxazepam from dosed feed (relative to gavage study at 50 mg/kg) was about 43%.