Notable pink excreta and severe myocardial suppression in superwarfarin (difethialone) intoxication.

Patients rarely consult physicians before developing coagulopathy or bleeding in most reported cases of superwarfarin intoxication. A 57-year-old woman ingested red-dyed pellets of anticoagulant rodenticide containing difethialone and warfarin as well as tablets of nitrazepam. Although she presented to the hospital in a comatose state, notable pink-colored excreta hinted at the consumption of anticoagulant rodenticide, which led to the early diagnosis of superwarfarin intoxication. Supplementation of large doses of intravenous and oral vitamin K successfully prevented coagulopathy and bleeding. On the other hand, temporary and reversible myocardial suppression was extremely severe, and required the introduction of percutaneous cardiopulmonary support.

Sexual assault under benzodiazepine submission in a Paris suburb.

Sexual assaults under benzodiazepine submission have been described, since use of benzodiazepine enables non consensual sexual activity but rarely fully reported. An accurate evaluation of the phenomenon has seemed interesting. Files of 23 adult males and females examined at the Emergency Forensic Unit of an University Teaching Hospital near Paris were reviewed. All the victims had complained from sexual assault under drug submission, in the years 1996 and 1997. A complete examination for sexual assault was realised linked to clinical examination of drug intoxication. Every victim of rape under drug submission was sampled for urine screening (mean delay of 17.5 h after sexual assault) and blood alcohol level quantification. Urine was screened for benzodiazepines, cocaine, opiates and cannabinoids with qualitative immunochromatographic test. Traumatic lesions of sexual penetration were retrieved in 10 victims and sperm in 5. Clinical signs of benzodiazepine intoxication were retrieved in 12 out of 23 victims. Urine benzodiazepine screening was positive, over the cut-off values (300 ng/mL)when sampled less than 20 h after the facts. In 6 out of 23 victims, drugs of abuse and alcohol were associated to benzodiazepines. A reinforced attention can be brought to the rape under drug submission including the need of a proper examination and samplings shortly after the alleged facts to ascertain the diagnosis and to help the victim facing the Justice inquiry.

Characterization of epididymal sperm motion and its correlation with stages of target cells in rats given alpha-chlorohydrin, cyclophosphamide or nitrazepam.

Epididymal sperm motion in rats was characterized by computer-aided sperm motion analysis (CASA) with its correlation to testicular lesions in the 2-week treatment study, using three compounds which are known to affect different stages of germ cells. Mature male rats were treated daily for 2 weeks with alpha-chlorohydrin (alpha-CH, 5 mg/kg), cyclophosphamide (CP, 20 mg/kg) or nitrazepam (NZ, 20, 40, 60 mg/kg). Changes in sperm motion were detected only in the alpha-CH and 60-mg/kg NZ-treated groups. Of the sperm motion parameters, velocity and amplitude of lateral head displacement (ALH) were concomitantly reduced in these two groups with good correlation. With respect to the distribution of the values in parameters, however, alpha-CH shifted the values down within a small range with high percentages of motile sperm, while NZ distributed them over a wide range with low percentages of motile sperm. CP treatment showed no histopathological changes in advanced germ cells, though it showed a decrease in the number of early germ cells. NZ treatment affected round and elongating spermatids (approximately step 14) at doses of 20 and 40 mg/kg, and affected also more advanced spermatids (approximately step 19) at the dose of 60 mg/kg. alpha-CH treatment did not affect testicular histopathology. These findings indicate that 60-mg/kg NZ treatment reduced sperm motion as a result of lesions affected in elongated spermatids and alpha-CH reduced it by direct effects on epididymal spermatozoa. The present study indicates that in addition to percentage of motile sperm, the velocity and ALH can be useful to detect the changes in sperm motion caused by different actions of NZ and alpha-CH, though each compound showed a distinct distribution pattern of these parameters.

Effects of psychotropic drugs on aldo-keto reductase activity in rat ovary and adrenal gland.

We investigated the effects of minor and major tranquilizers on ovarian and adrenal aldo-keto reductase activity towards five substrates in relation to ovulation in mature cycling rats. Nitrazepam (NZP) did not alter ovarian and adrenal weights or body weight, although ovulation was inhibited at 5 and 10 mg/kg. NZP decreased ovarian 13,14-dihydro-15-ketoprostaglandin F2 alpha (15KD-PGF2 alpha) and 4-benzoylpyridine (4BP) reducing activities. None of the doses of zopiclone (ZPC) influenced uterine and adrenal weights or body weight, but it increased ovarian weight at 10 mg/kg. No significant effects of ZPC on ovarian aldo-keto reductase activity were observed. NZP had inhibitory effects on adrenal aldo-keto reductase activity, whereas ZPC had a stimulatory effect. Chlorpromazine (CPZ) did not alter ovarian or adrenal weight, whereas the estrous cycles were abolished at 5 and 10 mg/kg. Reserpine (RSP) decreased ovarian weight and completely inhibited ovulation at 5 and 10 mg/kg, but it increased adrenal weight. Both CPZ and RSP decreased, dose dependently, ovarian aldo-keto reductase activity towards five substrates in agreement with the inhibition of ovulation. On the other hand, differences were found between the effects of CPZ and RSP on adrenal aldo-keto reductase activity. CPZ significantly increased 4BP reducing activity at 5 and 10 mg/kg, although no significant changes were observed in the other four reducing activities. RSP decreased 15KD-PGF2 alpha reducing activity in a dose-dependent manner, whereas the other four activities did not change.

Genetic toxicology of four commonly used benzodiazepines: a review.

Benzodiazepines are a group of drugs which have been extensively used for their activities as an anti-anxiety, sedative, muscle relaxant and anti-convulsant. Benzodiazepines at present are the most commonly prescribed drugs. Some of these drugs are teratogenic and also carcinogenic in experimental animals. The wide human exposure to this group of drugs throughout the world is of great concern for human health. In the present review, we have attempted to evaluate and update the mutagenic and genotoxic effects of four of the most commonly used benzodiazepines, i.e., chlordiazepoxide (CDZ), diazepam (DZ), nitrazepam (NZ) and oxazepam (OZ) based on available literature.

Male reproductive toxicity study of nitrazepam in rats.

The main focus of this study is the optimal administration period concerning toxic effects on male fertility in rats. To assess functional and morphological changes induced in the testis by nitrazepam, male rats were administered the drug at doses of 0, 20, 40 or 80 mg/kg during pre-mating periods of 2, 4 or 9 weeks and then the 2 weeks of mating. At the end of the administration period the animals were sacrificed and sperm number, motility, abnormalities and histopathological changes in the testis were examined. Decreases in testis weight, epididymis weight, number of sperm in the testis and sperm motility were observed in the 40 and 80 mg/kg sections of the 2, 4 and 9 week pre-mating treated groups. Mating with untreated females revealed no adverse effects on copulation rate in any group; however, a remarkable decrease in pregnancy rate was noted in the 80 mg/kg section of the 2, 4 and 9 week treated groups. On histological examination, various degrees of localized necrosis in the seminiferous epithelium and Leydig cell hyperplasia were observed in the testis. No clear changes were observed in the 20 mg/kg section of the 2 week pre-mating administration group, but at the 4 week time point, necrosis of spermatogenic cells began to appear. The primary morphological event was evident in spermatocytes with necrosis of the cytoplasm observed from 4 weeks after administration of nitrazepam, although sperm motility and sperm head counts were unaffected. From these findings, examination of sperm characteristics and histopathological changes in the testis are important parameters for evaluation of drugs inducing testicular damage. We conclude that a 4 week administration period is sufficient to detect effects of nitrazepam on male fertility.

Potential parameters of male reproductive toxicity: reproductive performance, histopathology and sperm evaluation in SD rats given nitrazepam.

The present study was designed to elucidate the correlation between findings from reproductive performance testing and those from histopathological examination of the testis and sperm analysis in rats given a benzodiazepine derivative, nitrazepam, for 2 and 4 weeks. The mechanisms of toxicological action of nitrazepam on the male reproductive organs were also investigated. Nitrazepam was given orally to Sprague-Dawley male rats (6-week-old) at a daily dose of 80 mg/kg for 2 weeks or at daily doses of 20, 40 or 80 mg/kg for 4 weeks. Treated males were mated to examine reproductive performance with untreated females after each dosing period, and after 4 and 9 week of recovery periods. Necropsy was performed for histopathological examination of the testis and epididymis and for sperm analysis after each dosing period and the final mating trial (total of 11 weeks recovery). In the findings from reproductive performance testing, significant decrease in the fertility index was observed in the 80 mg/kg group even after 2 weeks dosing and thereafter until 4 weeks recovery, though the mating index did not significantly differ from that of controls through the experiment. In the histopathological examination and sperm analysis, testicular signs of toxicity, decrease in number of sperm heads in the testis and increase in number of sperm with abnormal heads in the seminiferous tubules were noted in the 80 mg/kg group after 2 weeks dosing and in the 40 and 80 mg/kg groups after 4 weeks dosing. Concentrations of plasma testosterone and content of testis testosterone in nitrazepam-treated groups were not significantly different from those of controls. Plasma FSH concentration was significantly elevated in the 80 mg/kg group through the experiment, although significant elevation of plasma LH was observed only after 2 weeks dosing. These results indicate that histopathological examination is the most reliable approach to detect male reproductive adverse effects induced by nitrazepam rather than using parameters from mating trials. The four-week-dosing period is appropriate for their detection. Hypospermatogenesis induced by nitrazepam is suggested to be caused by direct action of nitrazepam on germ cells and/or Sertoli cells rather than by indirect action through inhibition of testosterone secretion.

Nitrazepam in patients with sleep apnoea: a double-blind placebo-controlled study.

We wanted to assess whether benzodiazepines worsen sleep apnoea, since their use in such patients has been controversial. Fourteen male patients with mild to moderate obstructive sleep apnoea were investigated in a placebo-controlled, double-blind study evaluating the influence of nitrazepam (NIT) on apnoea frequency and severity. Each patient was given oral nitrazepam 5 or 10 mg, or corresponding placebo, in a randomized order on three separate nights. Wash-out time was one week. A complete sleep study was undertaken at each study night. Eleven patients completed the study. Although there were individuals with marked variability in apnoea index between the three study nights, there was no significant change in apnoea index or minimum arterial oxygen saturation with any of the two nitrazepam dosages studied. Only 3 out of 11 patients had a higher apnoea index after both nitrazepam doses compared to placebo, and in these patients the increase in sleep-disordered breathing was of marginal clinical significance. Nitrazepam caused a modest increase in total sleep time and a decrease in rapid eye movement (REM) sleep. These results demonstrate that nitrazepam does not worsen sleep apnoea in patients with mild to moderate sleep apnoea. The previously reported sleep apnoea promoting effects of benzodiazepines may be restricted to a small subgroup of patients with sleep-disordered breathing.

Comparative developmental toxicity and metabolism of nitrazepam in rats and mice.

This study was designed to evaluate the developmental toxicity of nitrazepam (NZ) in Sprague-Dawley rats and ICR mice and to determine the metabolic factors which modulate susceptibility to the developmental effects of NZ. Rats were treated orally with a single dose of NZ at 300 mg/kg on Day 12 of gestation. Mice received one dose of 300 mg/kg NZ via gavage between Days 9 and 14 of gestation. NZ administration resulted in a significant incidence of malformations in rats, while no evidence of teratogenic action was observed in mice. Pronounced species differences in the metabolism of NZ were observed. In rats, 7-acetylaminonitrazepam (AANZ) was detected as the major metabolite in plasma and embryos, whereas in mice, only small amounts of this product were found. The rate of N-acetylation of 7-aminonitrazepam (ANZ) to AANZ was 8.5-fold greater in rat liver cytosol than that in mouse liver cytosol. In contrast, the rate of deacetylation of AANZ to ANZ was 9-fold greater in mouse liver microsomes than that in rat liver microsomes. The developmental effects of authentic metabolites of NZ were studied in the two species. A single oral administration of 300 mg/kg ANZ to pregnant animals produced a significant incidence of malformations in rats, but not in mice. On the other hand, AANZ was teratogenic in both species. These results suggest that the difference in the susceptibility to NZ-induced teratogenicity between rats and mice may be related to differences in the levels of N-acetyltransferase and deacetylase, and that AANZ may be involved in the teratogenic mechanism.

Involvement of the intestinal microflora in nitrazepam-induced teratogenicity in rats and its relationship to nitroreduction.

A study was undertaken to investigate the relationship between nitroreduction of nitrazepam and its teratogenic effects and the involvement of the intestinal microflora in Sprague-Dawley rats. Incubation of bacterial suspensions from rat cecal contents with nitrazepam resulted in extensive reduction to 7-aminonitrazepam. Rat liver homogenates also reduced nitrazepam but only under anaerobic conditions. Following oral administration of 300 mg/kg nitrazepam to pregnant rats, total excretion of reduced metabolites (7-aminonitrazepam and 7-acetylaminonitrazepam) in urine and feces accounted for approximately 30% of the administered dose. When antibiotics were administered to dams to deplete their intestinal microflora prior to administration to nitrazepam, the total excretion of the reduced metabolites in the urine and feces decreased to 2% of the dose. Nitroreductase activity of cecal contents was almost completely suppressed by antibiotic pretreatment, but the activity of liver homogenates was not significantly altered by the same treatment. The incidence of nitrazepam-induced malformations was markedly decreased by antibiotic pretreatment. These results suggest that the intestinal microflora plays an important role in the reductive metabolism of nitrazepam and that the teratogenicity of nitrazepam may be related to its nitroreduction by the microflora.

Teratogenic effects of nitrazepam in rats.

The teratogenicity of nitrazepam, a benzodiazepine-type drug, was investigated in Sprague-Dawley rats. Nitrazepam was administered to pregnant females at levels of 100, 200 or 300 mg/kg in a single dose on one of gestation days 10-14. Fetuses were examined for external and skeletal malformations on day 20 of gestation. Nitrazepam was found to produce various types of malformations, i.e., exencephaly, cleft palate, micrognathia, short or kinky tail and limb reduction defects. The predominant malformation was limb reduction defects, which were produced at high frequency by treatment on day 12 or 13 of gestation. Microscopic examination of limb buds revealed hemorrhage and mesenchymal cell necrosis at 24 hours after nitrazepam treatment.

In vivo and in vitro studies on fetal toxicity of benzodiazepines in rats.

In this study, we investigated whether the intensity in the embryotoxicity of benzodiazepines was related to maternal and fetal blood levels and also to their high binding to the fetoplacental structures in rats. Maternal and fetal levels of nitrazepam (NTZ) and nimetazepam were higher than the diazepam level after oral administration of these drugs (100 mg/kg). In vitro, the affinity of the drugs for the fetus corresponded to the drug levels in the maternal serum and fetus. In the embryo culture, NTZ had no direct teratogenicity but had embryocidal activity. On the contrary, the in vivo maternal serum treated with NTZ possessed both activities. From the above facts, and since there was no change in the in vivo embryotoxicity as a result of phenobarbital, it was suggested that the embryocidal activity of NTZ may be due to changes of maternal function depended upon the maternal serum.

Effects of repeated dose nitrazepam and lormetazepam on psychomotor performance in the elderly.

The daytime psychomotor performance of 12 healthy subjects of mean age 81.4 years was examined in a double-blind crossover study following single and repeated nightly doses of nitrazepam 5 mg, lormetazepam 1 mg, and placebo. Accuracy of performance on the Gibson spiral maze was unaffected by both drugs. On a reciprocal tapping task, speed was similarly unaffected. However, while neither drug significantly impaired accuracy on reciprocal tapping after the first dose, nitrazepam, but not lormetazepam, significantly reduced overall accuracy after seven consecutive doses, an effect consistent with drug accumulation.

Fetal toxicity of benzodiazepines in rats.

This study was undertaken to evaluate fetal toxicity of benzodiazepine derivatives, nitrazepam, oxazepam, nimetazepam, clonazepam, diazepam and chlordiazepoxide. In fetotoxicities induced by the oral administration of these six drugs (100 mg/kg), nitrazepam (NTZ) and nimetazepam caused a great damage to the fetus compared with other analogues. The fetotoxicity of NTZ was dose-dependent at a dose of 50 to 100 mg/kg p.o. and its peak toxicity was observed in the group treated from the 8th to 10th day of gestation. Maternal serum and whole fetus concentration of NTZ were higher than that of diazepam which showed weak toxicity. It was suggested that the intensity of fetotoxicity induced by benzodiazepines was related to the amount of drug in blood of dam.

Induction of sperm head abnormalities in mice by three tranquilizers.

Toxic effects of diazepam (DZ), chlordiazepoxide (CDZ) and nitrazepam (NZ) on the spermatozoa of mice have been studied at the end of 1, 3, 4, 6, 8 and 12 weeks after 15 days repeated treatment with a daily oral dose of 0.5 mg. Different types of abnormalities involving both shape and size of the sperm head were noticed. Qualitatively NZ produced the maximum number of abnormal types. The incidences of abnormal sperm heads were significantly high in all the test weeks in the NZ series, at weeks 1 and 6 after DZ treatment, and at weeks 3, 4 and 6 after CDZ treatment. All three drugs produced maximum effects at week 6. The varying effects induced by the three benzodiazepines seem to be due to their differential pharmacokinetics.

[In vivo cytogenetic studies of 10-chloro-11b-(2-fluorophenyl)-7-(2-hydroxyethyl)-2, 3, 5, 11b-tetrahydrooxazolo-(3, 2-d) (1, 4) benzodiazepine-6(7H)-one (MS-4101) on rat bone marrow cells (author's transl)].

Chromosome aberrations induced by MS-4101, diazepam, and nitrazepam were studied in Sprague-Dawley rats. Female rats were administered orally via stomach tube daily doses of 200, 500 and 2000 mg/kg of MS-4101, 200 and 500mg/kg of diazepam, 40 and 200mg/kg of nitrazepam and 20 and 50 mg/kg of cylcophosphamide as the positive control for one, 5 and 10 days. In this study, we used bone marrow cells derived from rat femur at 6 or 24 hours after the last drug administration. No significant chromosome aberrations were seen in the treated groups with MS-4101, diazepam, and nitrazepam when compared with the nontreated control. The increase of structural aberration on gap and break was observed in the group of cyclophosphamide as the positive control. These findings indicate that MS-4101, and diazepam and nitrazepam as similar comparative drugs, do not have the potential of induction of chromosome aberrations in rat bone marrow cells.

[Characteristics of the development of tolerance to nitrazepam and phenobarbital under experimental conditions]. / Osobennosti razvitiia tolerantnosti k nitrazapamu i fenobarbitalu v eksperimente.

Despite the similarity of the nitrazepam and phenobarbital spectra it was found that with their single usage the nature of tolerance for these substances differs quite substantially. With long-term introduction of nitrazepam to mice and rats in invariable and increasing doses there is seen a development of tolerance in the sense of the muscle relaxant and anticonvulsive action and in lethality without any marked attenuation of the tranquillizing effect. In contradistinction to this a long-term administration of phenobarbital is followed by a diminution of its tranquillizing action and by a slowly proceeding reduction of muscle relaxing manifestations. Crosswise tolerance among nitrazepam, diazepam and phenobarbital is marked but feebly. It is presumed that one of the possible mechanisms responsible for the nitrazepam tolerance in mice and rats may be a change in metabolic transformations and redistribution of metabolites.