New theoretical investigation of mechanism, kinetics, and toxicity in the degradation of dimetridazole and ornidazole by hydroxyl radicals in aqueous phase.

Dimetridazole (DMZ) and ornidazole (ONZ) have been widely used to treat anaerobic and protozoal infections. The residues of DMZ/ONZ persist in the water environment. The mechanisms and kinetics of hydroxyl-initiated oxidation, the primary DMZ/ONZ degradation method, were evaluated by quantum chemical methods.·OH-induced degradation of DMZ and ONZ shared many mechanistic and kinetic characteristics. The most feasible degradation pathway involved forming OH-imidazole adducts and NO2. The OH-imidazole adducts were subsequently degraded into double·OH imidazole intermediates. The rate coefficients for·OH degradation of DMZ and ONZ were 4.32 × 109 M-1 s-1 and 4.42 × 109 M-1 s-1 at 298 K, respectively. The lifetimes of DMZ and ONZ treated with·OH at concentrations of 10-9-10-18 mol L-1 at 298 K were τDMZ = 0.231-2.31 × 108 s and τONZ = 0.226-2.26 × 108 s, respectively. Toxicity assessment showed that the first degradation products of DMZ and ONZ exhibited enhanced aquatic toxicity, whereas most of the secondary degradation products were not harmful to aquatic organisms. Some of transformation products were still developmental toxicant or mutagenicity positive.

[Mechanisms of Huanshao Capsules protecting the reproductive function in rats with ornidazole-induced asthenozoospermia].

OBJECTIVE: To explore the possible mechanism of Huanshao Capsules (HSC) protecting the reproductive function in rats with ornidazole-induced asthenozoospermia (AZS). METHODS: Forty SD male rats were randomly divided into four groups of equal number, blank control, AZS model control, HSC and L-carnitine (LC) intervention. The AZS model was established in the latter three groups of rats by intragastrical administration of ornidazole at 400 mg/kg/d for 28 days, and meanwhile the animals in the HSC and LC groups were treated by gavage of HSC at 0.31 g/kg/d and LC at 100 mg/kg/d, respectively. Then, all the rats were killed for examination of the LC content, sperm concentration, sperm motility and expression of OCTN2 mRNA in the epididymis and observation of the histopathological changes in the testis tissue. RESULTS: Compared with the AZS model controls, the rats in the HSC and LC groups showed significantly increased LC content (2 880.3 vs 6 366.5 and 6 934.7 mg/L, P < 0.01), sperm concentration (ï¼»34.58 ± 10.25ï¼½ vs ï¼»46.19 ± 14.23ï¼½ and ï¼»42.25 ± 6.11ï¼½ ×106/ml, P < 0.01), sperm motility (ï¼»42.59 ± 7.54ï¼½% vs ï¼»61.34 ± 7.98ï¼½% and ï¼»61.34 ± 7.98ï¼½%, P < 0.01) and expression of OCTN2 mRNA in the epididymis (26.07% vs 27.26% and 27.15%, P < 0.01). The animals of the HSC group exhibited a higher comparability than those of the LC group to the blank controls in the morphology, arrangement and activity of spermatogenic cells. CONCLUSIONS: HSC can protect the reproductive function and improve sperm concentration and motility in the model rats with ornidazole-induced AZS, which may be associated with its abilities of up-regulating the expression of OCTN2 mRNA and increasing the LC content in the epididymis.

DJ-1 deficiency causes metabolic abnormality in ornidazole-induced asthenozoospermia.

Asthenozoospermia (AS), defined as low-motility spermatozoa in the ejaculate, is a frequent cause of human male infertility. DJ-1 (also known as PARK7), a protein highly associated with male sterility, binds to the mitochondrial complex I subunit to protect mitochondrial function. However, its involvement in spermatogenesis has not been fully elucidated. Previously, the levels of DJ-1 were shown to be significantly decreased in testicular tissues of rats with ornidazole (ORN)-induced AS. Here, we used a rat model to investigate the localization and expression levels of DJ-1 and its interacting NDUFS3 and NDUFA4 mitochondrial complex I subunits, as well as AS-induced metabolic alterations in testicular tissues. ORN significantly reduced the levels of DJ-1 in the nucleus of secondary spermatocytes, while increasing the expression of NDUFS3 in the cytoplasm of primary spermatocytes. Further, NDUFA4 showed higher expression after treatment with ORN. The principal ORN-induced changes in metabolic small molecules related to the accumulation of glucose, glutamine, and N-acetyl aspartate, enhancement of purine pathway, increase of the phosphatidic acid (PA) (18:0/18:1), phosphatidylethanolamine (PE) (16:0/18:1), and PA (18:0/20:4) lipid metabolites, and imbalance in the concentrations of Na+ and K+. However, we did not observe any abnormalities of certain small metabolic molecules and metal ions in semen samples from patients with AS. In conclusion, these results suggest that DJ-1 deficiency in testicular tissues might be closely related to the localization of NDUFS3 and content of NDUFA4, thus causing abnormalities in the mitochondrial energy metabolism and multiple other metabolic pathways.

Biomarkers of genotoxicity and genomic instability in a non-human primate, Cebus libidinosus (Cebidae, Platyrrhini), exposed to nitroimidazole derivatives.

The genotoxicity of two nitroimidazole derivatives, ornidazole (ONZ) and metronidazole (MTZ) in the peripheral blood lymphocytes of Cebus libidinosus (CLI) (Primates, Cebidae) was assessed. Endpoints measured included sister chromatid exchange (SCE) frequency, cell proliferation kinetics (CPK), replication index (RI), mitotic index (MI), and damage incidence in or near CLI heterochromatin regions. MI and SCE values following ONZ or MTZ treatments were significantly different (p<0.001) from control. SCE frequency per chromosome was not proportional to chromosome length. The chromosomes most affected for SCE were 1, 2, 4, 6, 11-13, 17, and 18, many of which possess interstitial or terminal heterochromatin. In the CLI genome, chromosomes 11 and 17 showed higher susceptibility to damage RI was the only biomarker that did not show statistically significant differences between control and treated cultures. C. libidinosus bands 11q1.4 and 11q1.5 may be hot-spots in the context of nitroimidazole exposure.

The role of C-type natriuretic peptide in rat testes during spermatogenesis.

C-type natriuretic peptide (CNP) is a 22-amino acid peptide and act as a local paracrine or autocrine regulator. There is growing evidence that CNP is involved in male reproductive processes. To investigate the role of CNP during spermatogenesis, we measured the mRNA expression of CNP and its specific membrane-bound natriuretic peptide receptor-B (NPR-B) using real-time RT-PCR in the testes of normal rats on different postnatal days. After that spermatogenesis dysfunction model induced by ornidazole was established with the aim to study the correlation of CNP with spermatogenic dysfunction. Then, Sertoli cells from 18- to 22-day-old healthy male rats were cultured in the presence of different CNP concentrations (1×10(-6), 1×10(-7) and 1×10(-8) mol l(-1)), and the mRNA expression levels of androgen-binding protein, inhibin B and transferrin were examined at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h. During the postnatal development of rat testes, the highest mRNA expression levels of CNP and NPR-B were found at postnatal D(0), and the levels then declined gradually, with a second CNP peak at postnatal D(35). In the ornidazole-induced infertile rat testes, CNP gene expression was lower than in the uninduced rats (P<0.05), while NPR-B gene expression was greater (P<0.05). In cultured Sertoli cells, supplementation with CNP stimulated the gene expression of androgen-binding protein/inhibin B/transferrin, particularly at 12 h, and 1×10(-7) mol l(-1) CNP had the highest upregulation effect. The gene expression levels of CNP/NPR-B in rat testes at different postnatal stages and in infertile rat testes indicated that CNP may participate in the physiology and/or pathology related to spermatogenesis. Moreover, CNP regulated endocrine function in Sertoli cells. Taken together, these results showed that CNP is closely tied to spermatogenesis.

Nitroimidazole derivatives: non-randomness sister chromatid exchanges in human peripheral blood lymphocytes.

Nitroimidazole derivatives exhibited genotoxic effect in different experimental conditions. This study focuses on an evaluation of possible genomic targets, at a chromosomal level, of two 5-nitroimidazoles (ornidazole and metronidazole) using the in vitro human peripheral blood culture as experimental system. We observed that both derivatives showed a decrease in mitotic index (MI) (P < 0.001), an increase in sister chromatid exchanges (SCE) frequency (P < 0.001) and no modifications in cellular proliferation kinetics (CPK). As a null hypothesis we considered the assumption that larger chromosomes should harbor more SCE, which was viewed using a novel sequential G-band (400 band resolution)/SCE technique. The analysis showed highly significant chi square values (P < 0.001), indicating that SCE frequency per chromosome is not proportional to chromosome length. SCE could be considered an instability indicator due to the high correlation between SCEs in certain chromosomal bands and the exposure to nitroimidazole derivatives.

Antimicrobial drug ornidazole inhibits hamster sperm capacitation, in vitro.

To be fertilization competent, spermatozoa undergo a series of changes in the female reproductive tract collectively referred to as capacitation. In an attempt to understand, if ornidazole, a known anti-fertility drug, adversely affects sperm functions by targeting capacitation, we designed experiments to study the influence of this drug on hyperactivation (HA), capacitation-associated protein tyrosine phosphorylation (pY) and the acrosome reaction (AR). Addition of ornidazole at 0 h, inhibited the onset of HA and total pY in a dose dependent manner. However, when ornidazole was added at 3.5h, severe effects were still seen on HA and pY of high molecular weight proteins but, pY of lower M(r) proteins (50-56 kDa) was affected only marginally. Further, lower doses of ornidazole (5 and 10 mM) had greater inhibitory effect when added at 0 h, while addition of ornidazole at 3.5 h required higher doses of ornidazole (25 mM) to cause significant inhibition of acrosome reaction. Collectively, through in vitro studies, we demonstrate that ornidazole affects the onset and progression of hamster sperm hyperactivation, capacitation associated protein tyrosine phosphorylation and acrosome reaction, and the severity depends on the dose (5, 10 or 25 mM) and the time of addition (0 or 3.5 h) of the drug to the spermatozoa.

Cytogenetic evaluation of two nitroimidazole derivatives.

5-Nitroimidazoles are a well-established group of antiprotozoal and antibacterial agents. Thanks to their antimicrobial activity these chemotherapeutic agents inhibit the growth of both anaerobic bacteria and certain anaerobic protozoa such as Trichomonas vaginalis, Entamoeba histolytica and Giardia lamblia. The aim of the present study is to achieve a precise characterization of the genotoxic activity of these compounds and to establish the value of cytogenetic assays in order to determine the effect of these drugs, at therapeutic doses, to settle an improved risk assessment. Two nitroimidazole were studied, metronidazole and ornidazole, at four different concentrations (0.1, 1, 10 and 50 microg/ml of peripheral blood lymphocyte culture). Endpoints analyzed included: mitotic index (MI), replication index (RI), sister chromatid exchange (SCE) and chromosomal aberrations (CA). An analysis of variance test (ANOVA) was performed to evaluate the results. A significant decrease (P<0.0001) in MI as well as an increase in SCE (P<0.0001) and CA (0.0001) frequencies for both drugs was observed. No modifications in RI were found. The results suggest a genotoxic and cytotoxic effect of MTZ and ONZ in human peripheral blood cultures in vitro.

DJ-1, a target protein for an endocrine disrupter, participates in the fertilization in mice.

DJ-1 was first identified as an activated ras-dependent oncogene product and was later also found to be an infertility-related protein affected by sperm toxicants such as ornidazole (OR) and epichlorohydrin. These findings suggest that DJ-1 has functions in both somatic cells and sperm. In this study, to determine the relationship between DJ-1 and an endocrine disrupter and to determine the functions of DJ-1 in sperm, in vitro fertilization experiments were carried out using eggs and sperm extracted from mice that had or had not been treated with OR. We found that the amount of DJ-1 in sperm and the efficiency of fertilization decreased with the increasing dose of OR to which the mice were exposed. The addition of an anti-mouse DJ-1 serum to sperm solution before the in vitro fertilization reaction with eggs resulted in a decrease in the efficiency of fertilization to about one-third of that when pre-immune serum was added to sperm solution, indicating that DJ-1 participates in the fertilization.

DNA single strand breaks in peripheral blood lymphocytes induced by three nitroimidazole derivatives.

Tinidazole (TNZ), ornidazole (ONZ) and metronidazole (MTZ) are antiparasitic drugs (nitroimidazole derivatives) that have proven to be effective against Trichomonas vaginalis, Entoamoeba histolytica, Giardia lamblia and Helicobacter pylori. The reduction of the nitro group and the generation of short-lived reactive intermediates are the basis of its parasiticidal activity. This reduction is associated with its mutagenic activity in bacteria, although in mammalian cells DNA damage seems to be related to the production of reactive oxygen species (ROS). Using alkaline single cell electrophoresis, a significant increase in single strand breaks and alkali labile sites in human peripheral blood lymphocytes (PBL) exposed to MTZ, ONZ and TNZ at 10, 100 and 500 microg/ml is observed. MTZ causes less damage, especially at higher concentrations, when compared with TNZ, the most harmful of the drugs tested. These findings suggest that primary damage is induced under aerobic conditions and confirms that these nitroimidazoles are DNA damaging agents.

Toxicity of ornidazole and its analogues to rat spermatozoa as reflected in motility parameters.

Ornidazole, a 5-nitro-imidazole derivative, has contraceptive properties in rats. As some ornidazole passes through the body unmetabolized after administration, the aim of this study was to investigate if ornidazole itself has a direct effect on sperm motility and whether these effects are limited or potentiated by the epididymal epithelium or structural changes to the molecule. Cauda epididymal spermatozoa or cauda epididymal tubules were incubated with ornidazole or ornidazole analogues, and motility parameters were subsequently measured by means of a computer-assisted sperm analysis (CASA) system. Incubation of spermatozoa in 2.5 mmol/L ornidazole for 4 h reduced their motility significantly, whereas incubation of epididymal tubules for 8 h in 10 mmol/L ornidazole was required to alter the velocity parameters of the enclosed spermatozoa upon release, suggesting that extratubular non-metabolized ornidazole can participate in inhibiting the motility in vivo. The in vitro toxicity of ornidazole derivatives depends on the halogen present and on the position of the nitro-group. The putatively inactive (R)- and the active (S)-ornidazole exhibited equivalent depression of sperm motility by direct incubation. This observation, and the differences between the in vitro and the in vivo efficacies of various ornidazole analogues, indicates distinct mechanisms of motility inhibition in the two experimental systems.

Effects of three male reproductive toxicants on rat cauda epididymal sperm motion.

The sensitivity of the CellSoft computer-assisted sperm analysis (CASA) system to detect changes in rat sperm motion was evaluated. CASA motion endpoints were measured in cauda epididymal sperm from Long-Evans rats treated with each of three known male reproductive toxicants reported to affect the epididymis and epididymal sperm motility: alpha-chlorohydrin, ornidazole, and trimethylphosphate. Significant changes in endpoints describing sperm swimming vigor (curvilinear velocity and straight-line velocity) and pattern (linearity and amplitude of lateral head displacement) were observed for rats dosed with each agent when evaluations included mean values and other statistical parameters (i.e., percentiles and distributional shape). alpha-Chlorohydrin (ACH) treatment (10 mg/kg/day; 8 days) resulted in reductions in the mean percentage of motile sperm, curvilinear velocity (VCL), straight-line velocity (VSL), lateral head displacement (ALH), and linearity (LIN). Treatment with ornidazole (ONZ) (200 mg/kg/day/14 days) reduced the percentage of motile sperm. Mean VCL, VSL, and ALH were reduced by 400 mg ONZ/kg/day treatment. Trimethylphosphate (TMP) treatment led to (a) a reduction in the 75th and 90th percentiles for ALH (100 mg TMP/kg/day; 5 days) (P < or = 0.04), (b) a reduction in VCL, VSL, and ALH (250 mg TMP/kg/day), (c) a reduction in the percentage of motile cells and in the 10th and 25th percentiles for VSL (600 mg TMP/kg/day), and (d) increases in the 90th percentile for VSL, in the mean, 75th, and 90th percentiles for VCL, and in the 75th and 90th percentiles for ALH (600 mg TMP/kg/day). The general utility of these analytic approaches in reproductive toxicology studies was demonstrated in the observations of effects at or below dose levels previously reported.

Reproduction studies in rats treated with ornidazole.

Reproduction studies were performed with ornidazole, a compound with trichomonacidal activity. Male rats were treated for 61 days prior to mating and female rats were treated for 2 weeks prior to mating and throughout gestation and lactation at doses of 0 (control), 25, 100, and 400 mg of ornidazole/kg/day. A decrease in the pregnancy rate was observed in high-dose rats without altered mating performance. Crossover matings between high-dose treated and control male and female rats showed that male but not female fertility was affected and that the effect on fertility was reversible within several days after the cessation of treatment. Testicular and epididymal weights were not altered in treated male rats. Histopathological examination revealed that spermatogenesis and the testes were normal and that the epididymides of treated male rats contained normal appearing sperm. It is concluded that ornidazole, at high dosages, produces infertility in the male rat; however, unlike many other 5-nitroimidazole compounds which are reported to inhibit spermatogenesis, no effect on spermatogenesis was observed under the conditions of these studies. This in conjunction with the rapid reversibility of infertility suggests that the mode of action of ornidazole involves a rapidly reversible effect on epididymal sperm function.

The effect of ornidazole on fertility and epididymal sperm function in rats.

A comprehensive study of male fertility and sperm production and function was performed in 20 control and 20 rats treated with ornidazole, a compound with trichomonacidal activity. Rats were treated for 4 weeks at dosages of 0 (control) and 400 mg/kg/day of ornidazole during which fertility was assessed by weekly matings. Testicular sperm production and epididymal sperm function were assessed in one-half of the rats while the reversibility of effects after a 2-week recovery period was assessed in the remaining half. Male rats treated with ornidazole were infertile during the second week of treatment. After 4 weeks of treatment, testicular and epididymal weights, testicular spermatid counts, epididymal sperm reserves, sperm morphology, and sperm viability were similar in treated and control rats. A quantitative assessment of epididymal sperm motility using a dark-field photomicroscope with a stroboscopic light source revealed that ornidazole markedly inhibited sperm motility. Although the percentage of nonmotile sperm was not substantially increased in treated rats, the vigor of tail movement was markedly decreased which resulted in decreased sperm velocity. Restoration of fertility and normal sperm motility and velocity were observed in the group of recovery rats assessed 2 weeks after the cessation of ornidazole treatment. It is concluded that ornidazole, at a high dosage of 400 mg/kg/day, produces infertility in male rats by inhibiting epididymal sperm motility in terms of decreased sperm velocity. These effects are rapidly reversible after the cessation of treatment.