Oxidative inactivation of the endogenous antioxidant protein DJ-1 by the food contaminants 3-MCPD and 2-MCPD.

3-Chloro-1,2-propanediol (3-MCPD) and 2-chloro-1,3-propanediol (2-MCPD) are heat-induced food contaminants being present either as free substances or as fatty acid esters in numerous foods. 3-MCPD was classified to be possibly carcinogenic to humans (category 2B) with kidney and testis being the primary target organs according to animal studies. A previous 28-day oral feeding study with rats revealed that the endogenous antioxidant protein DJ-1 was strongly deregulated at the protein level in kidney, liver, and testis of the experimental animals that had been treated either with 3-MCPD, 2-MCPD or their dipalmitate esters. Here we show that this deregulation is due to the oxidation of a conserved, redox-active cysteine residue (Cys106) of DJ-1 to a cysteine sulfonic acid which is equivalent to loss of function of DJ-1. Irreversible oxidation of DJ-1 is associated with a number of oxidative stress-related diseases such as Parkinson, cancer, and type II diabetes. It is assumed that 3-MCPD or 2-MCPD do not directly oxidize DJ-1, but that these substances induce the formation of reactive oxygen species (ROS) which in turn trigger DJ-1 oxidation. The implications of 3-MCPD/2-MCPD-mediated ROS formation in vivo for the ongoing risk assessment of these compounds as well as the potential of oxidized DJ-1 to serve as a novel effect biomarker for 3-MCPD/2-MCPD toxicity are being discussed.

Comparative proteomic analysis of 2-MCPD- and 3-MCPD-induced heart toxicity in the rat.

The chlorinated propanols 2- and 3-monochloropropanediol (MCPD), and their fatty acid esters have gained public attention due to their frequent occurrence as heat-induced food contaminants. Toxic properties of 3-MCPD in kidney and testis have extensively been characterized. Other 3-MCPD target organs include heart and liver, while 2-MCPD toxicity has been observed in striated muscle, heart, kidney, and liver. Inhibition of glycolysis appears to be important in 3-MCPD toxicity, whereas mechanisms of 2-MCPD toxicity are still unknown. It is thus not clear whether toxicity by the two isomeric compounds is dependent on similar or dissimilar modes of action. A 28-day oral feeding study in rats was conducted using daily non-toxic doses of 2-MCPD or 3-MCPD [10 mg/kg body weight], or an equimolar (53 mg/kg body weight) or a lower (13.3 mg/kg body weight) dose of 2-MCPD dipalmitate. Comprehensive comparative proteomic analyses of substance-induced alterations in the common target organ heart revealed striking similarities between effects induced by 2-MCPD and its dipalmitate ester, whereas the degree of effect overlap between 2-MCPD and 3-MCPD was much less. The present data demonstrate that even if exerting effects in the same organ and targeting similar metabolic networks, profound differences between molecular effects of 2-MCPD and 3-MCPD exist thus warranting the necessity of separate risk assessment for the two substances. This study for the first time provides molecular insight into molecular details of 2-MCPD toxicity. Furthermore, for the first time, molecular data on 3-MCPD toxicity in the heart are presented.

Twenty-six-week oral carcinogenicity study of 3-monochloropropane-1,2-diol in CB6F1-rasH2 transgenic mice.

The carcinogenic potential of 3-monochloro-1,2-propanediol (3-MCPD) was evaluated in a short-term carcinogenicity testing study using CB6F1 rasH2-Tg (rasH2-Tg) mice. 3-MCPD is found in many foods and food ingredients as a result of storage or processing and is regarded as a carcinogen since it is known to induce Leydig cell and kidney tumors in rats. Male and female rasH2-Tg mice were administered 3-MCPD once daily by oral gavage at doses of 0, 10, 20, and 40 mg/kg body weight (bw) per day for 26 weeks. As a positive control, N-methyl-N-nitrosourea (MNU) was administered as a single intraperitoneal injection (75 mg/kg). In 3-MCPD-treated mice, there was no increase in the incidence of neoplastic lesions compared to the incidence in vehicle control mice. However, 3-MCPD treatment resulted in an increased incidence of tubular basophilia in the kidneys and germ cell degeneration in the testes, with degenerative germ cell debris in the epididymides of males at 20 and 40 mg/kg bw per day. In 3-MCPD-treated females, vacuolation of the brain and spinal cord was observed at 40 mg/kg bw per day; however, only one incidence of vacuolation was observed in males. Forestomach and cutaneous papilloma and/or carcinoma and lymphoma were observed in most rasH2 mice receiving MNU treatment. We concluded that 3-MCPD did not show carcinogenic potential in the present study using rasH2-Tg mice. The findings of this study suggest that the carcinogenic potential of 3-MCPD is species specific.

2-Chloro-1,3-propanediol (2-MCPD) and its fatty acid esters: cytotoxicity, metabolism, and transport by human intestinal Caco-2 cells.

The food contaminants 3-chloro-1,2-propanediol (3-MCPD) and 3-MCPD fatty acid esters have attracted considerable attention in the past few years due to their toxic properties and their occurrence in numerous foods. Recently, significant amounts of the isomeric compounds 2-chloro-1,3-propanediol (2-MCPD) fatty acid esters have been detected in refined oils. Beside the interrogation which toxic effects might be related to the core compound 2-MCPD, the key question from the risk assessment perspective is again-as it was discussed for 3-MCPD fatty acid esters before-to which degree these esters are hydrolyzed in the gut, thereby releasing free 2-MCPD. Here, we show that free 2-MCPD but not 2-MCPD fatty acid esters were able to cross a monolayer of differentiated Caco-2 cells as an in vitro model for the human intestinal barrier. Instead, the esters were hydrolyzed by the cells, thereby releasing free 2-MCPD which was neither absorbed nor metabolized by the cells. Cytotoxicity assays revealed that free 2-MCPD as well as free 3-MCPD was not toxic to Caco-2 cells up to a level of 1 mM, whereas cellular viability was slightly decreased in the presence of a few 2-MCPD and 3-MCPD fatty acid esters at concentrations above 10 µM. The observed cytotoxic effects correlated well with the induction of caspase activity and might be attributed to the induction of apoptosis by free fatty acids which were released from the esters in the presence of Caco-2 cells.

Orally administered glycidol and its fatty acid esters as well as 3-MCPD fatty acid esters are metabolized to 3-MCPD in the F344 rat.

IARC has classified glycidol and 3-monochloropropane-1,2-diol (3-MCPD) as group 2A and 2B, respectively. Their esters are generated in foodstuffs during processing and there are concerns that they may be hydrolyzed to the carcinogenic forms in vivo. Thus, we conducted two studies. In the first, we administered glycidol and 3-MCPD and associated esters (glycidol oleate: GO, glycidol linoleate: GL, 3-MCPD dipalmitate: CDP, 3-MCPD monopalmitate: CMP, 3-MCPD dioleate: CDO) to male F344 rats by single oral gavage. After 30 min, 3-MCPD was detected in serum from all groups. Glycidol was detected in serum from the rats given glycidol or GL and CDP and CDO in serum from rats given these compounds. In the second, we examined if metabolism occurs on simple reaction with rat intestinal contents (gastric, duodenal and cecal contents) from male F344 gpt delta rats. Newly produced 3-MCPD was detected in all gut contents incubated with the three 3-MCPD fatty acid esters and in gastric and duodenal contents incubated with glycidol and in duodenal and cecal contents incubated with GO. Although our observation was performed at 1 time point, the results showed that not only 3-MCPD esters but also glycidol and glycidol esters are metabolized into 3-MCPD in the rat.

Nephrotoxicity evaluation of 3-monochloropropane-1,2-diol exposure in Sprague-Dawley rats using data-independent acquisition-based quantitative proteomics analysis.

3-Monochloropropane-1,2-diol (3-MCPD), as a heat-induced food process contaminant, possesses strongly toxic effect on kidney. The present study focuses on characterizing the proteome and clarifying the underlying molecular regulatory mechanisms in a model of kidney injury in rats treated with 3-MCPD. Data-independent acquisition (DIA)-mass spectrometry (MS) based proteomics was used to identify dysregulated proteins in kidney tissues of Sprague-Dawley (SD) rats treated with 30 mg/kg/day 3-MCPD by gavage for 28 days. It was found that a total of 975 proteins were deregulated after 3-MCPD treatment. Bioinformatic analyses revealed that several enzymes related to the metabolisms of amino acid, lipid and carbohydrate in endogenous metabolism were altered in response to 3-MCPD treatment. Moreover, some proteins involved in these pathways were also changed, mainly including oxidative stress, oxidative phosphorylation, apoptosis and autophagy. Our study unravels the vital roles of loss of mitochondrial homeostasis and function and cell death pathways in the development of renal damage induced by 3-MCPD, which provides further valuable insights into the initiation and resolution of 3-MCPD nephrotoxicity. The proposed DIA-MS workflow not only provides a choice for proteomic analysis in toxicological research, but also provides a more comprehensive understanding of the molecular mechanisms of nephrotoxicity induced by toxins.

Metabolites of 2- and 3-Monochloropropanediol (2- and 3-MCPD) in Humans: Urinary Excretion of 2-Chlorohydracrylic Acid and 3-Chlorolactic Acid after Controlled Exposure to a Single High Dose of Fatty Acid Esters of 2- and 3-MCPD.

SCOPE: Fatty acid esters of 2-monochloropropane-1,3-diol (2-MCPD) and 3-monochloropropane-1,2-diol (3-MCPD) are formed during the deodorization of vegetable oils. After lipase-catalyzed hydrolysis in the intestine, 2- and 3-MCPD are absorbed, but their ensuing human metabolism is unknown. METHODS AND RESULTS: The compounds 2-chlorohydracrylic acid (2-ClHA) and 3-chlorolactic acid (3-ClLA) resulting from oxidative metabolism of 2-MCPD and 3-MCPD, respectively, are identified and quantified in human urine samples. An exposure study with 12 adults is conducted to determine the urinary excretion of 2-ClHA and 3-ClLA. The participants eat 12 g of hazelnut oil containing 24.2 mg kg-1 2-MCPD and 54.5 mg kg-1 3-MCPD in the form of fatty acid esters. Average daily amounts of "background" excretion before the exposure are 69 nmol 2-ClHA and 3.0 nmol 3-ClLA. The additional mean excretion due to the uptake of the hazelnut oil amounts to 893 nmol 2-ClHA (34.0% of the 2-MCPD dose) and 16.4 nmol 3-ClLA (0.28% of the 3-MPCD dose). CONCLUSIONS: The products of oxidative metabolism of 2- and 3-MCPD, 2-ClHA, and 3-ClLA, are described for the first time in humans. Due to the lack of specificity, the metabolites may not be used as exposure biomarkers to low doses of bound 2- and 3-MCPD, respectively.

Developmental toxic potential of 1,3-dichloro-2-propanol in Sprague-Dawley rats.

This study investigated the potential adverse effects of 1,3-dichloro-2-propanol (1,3-DCP) on pregnant dams and the embryo-fetal development after maternal exposure on gestational days (GD) 6 through 19 in Sprague-Dawley rats. The test chemical was administered to pregnant rats by gavage at dose levels of 0, 10, 30, and 90mg/kg per day (n=10 for each group). All dams underwent Caesarean sections on GD 20, and their fetuses were examined for morphological abnormalities. Maternal toxicity was noted at 90mg/kg/day. Manifestations of toxicity included clinical signs of illness, lower body weight gain, decreased food intake, and increases in the weight of the adrenal glands and the liver. Developmental toxic effects including decreases in fetal body weight and increases in visceral and skeletal variations also occurred at the highest dose. At 30mg/kg, only a minimal maternal toxicity, including a decrease in maternal food intake and an increase in the liver weight, was observed. No adverse maternal or developmental effects were observed at 10mg/kg/day. These results revealed that a 14-day repeated oral dose of 1,3-DCP was minimally embryotoxic but not teratogenic at a maternal toxic dose (90mg/kg/day), and was not embryotoxic at a minimally maternal toxic dose (30mg/kg/day) in rats. Because the developmental toxicity of 1,3-DCP was observed only in the presence of maternal toxicity, it is concluded that the developmental findings observed in the present study are secondary effects to maternal toxicity. Under these experimental conditions, the no-observed-adverse-effect level of 1,3-DCP is considered to be 10mg/kg/day for dams and 30mg/kg/day for embryo-fetal development.

Protective effects of apigenin against 3-MCPD-induced renal injury in rat.

Apigenin (API) is a kind of important flavonoid present in temperate and tropical fruit and vegetables, especially the celery. It exerts anticancer, anti-bacterial, anti-viral, anti-inflammatory, anti-oxidation properties. In the present study, the mechanism of protective action of apigenin on 3-chloro-1, 2-propanediol (3-MCPD)-induced renal injury was investigated in rat. Sprague-Dawley (SD) rats were divided into five groups: control group (ultrapure water treated), CMC group (sodium carboxymethylcellulose treated), 3-MCPD treatment group (30 mg/kg body weight/day), 3-MCPD plus API co-treatment group (20, 40 mg/kg body weight/day). The results showed that API significantly reduced renal function markers, serum creatinine and urea nitrogen content. Besides, the renal tissue lesion in 3-MCPD treatment group was restored by API to some extent. We indicated that API exerted renoprotective effect by modulating oxidative phosphorylation especially up-regulated the expressions of ATP6 and ATP8, re-establishing mitochondrial membrane potential (MMP), relieving the increase of Bax/Bcl2 ratio, reducing cytochrome c release, thus inhibiting the activation of Caspase 9 and Caspase 3. In conclusion, apigenin possesses excellent protective effect against 3-MCPD-induced renal injury by modulating mitochondria dependent Caspase cascade pathway.

Absorption, Distribution, Metabolism and Excretion of 3-MCPD 1-Monopalmitate after Oral Administration in Rats.

Fatty acid esters of monochloropropane 1,2-diol (3-MCPD) are processing-induced toxicants and have been detected in several food categories. This study investigated the absorption, distribution, metabolism, and excretion of 3-MCPD esters in Sprague-Dawley (SD) rats using 3-MCPD 1-monopalmitate as the probe compound. The kinetics of 3-MCPD 1-monopalmitate in plasma was investigated using SD rats, and the results indicated that 3-MCPD 1-monopalmitate was absorbed directly in vivo and metabolized. Its primary metabolites in the liver, kidney, testis, brain, plasma, and urine were tentatively identified and measured at 6, 12, 24, and 48 h after oral administration. Structures were proposed for eight metabolites. 3-MCPD 1-monopalmitate was converted to free 3-MCPD, which formed the phase II metabolites. All of the metabolites were chlorine-related chemical components; most of them existed in urine, reflecting the excretion pattern of 3-MCPD esters. Understanding the metabolism of 3-MCPD esters in vivo is critical for assessing their toxicities.

Measurement of micronuclei and internal dose in mice demonstrates that 3-monochloropropane-1,2-diol (3-MCPD) has no genotoxic potency in vivo.

In this study 3-monochloropropane-1,2-diol (3-MCPD), a compound that appears as contaminant in refined cooking oils, has been studied with regard to genotoxicity in vivo (mice) with simultaneous measurement of internal dose using state-of-the-art methodologies. Genotoxicity (chromosomal aberrations) was measured by flow cytometry with dual lasers as the frequency of micronuclei in erythrocytes in peripheral blood from BalbC mice intraperitoneally exposed to 3-MCPD (0, 50, 75, 100, 125 mg/kg). The internal doses of 3-MCPD in the mice were calculated from N-(2,3-dihydroxypropyl)-valine adducts to hemoglobin (Hb), quantified at very low levels by high-resolution mass spectrometry. Convincing evidence for absence of genotoxic potency in correlation to measured internal doses in the mice was demonstrated, despite relatively high administered doses of 3-MCPD. The results are discussed in relation to another food contaminant that is formed as ester in parallel to 3-MCPD esters in oil processing, i.e. glycidol, which has been studied previously by us in a similar experimental setup. Glycidol has been shown to be genotoxic, and in addition to have ca. 1000 times higher rate of adduct formation compared to that observed for 3-MCPD. The conclusion is that at simultaneous exposure to 3-MCPD and glycidol the concern about genotoxicity would be glycidol.

Analysis of 3-MCPD- and 3-MCPD dipalmitate-induced proteomic changes in rat liver.

3-Monochloropropane-1,2-diol (3-MCPD) and 3-MCPD fatty acid esters are process contaminants in foodstuff which are generated during thermal treatment. Long-term exposure to 3-MCPD or 3-MCPD esters causes toxicity especially in kidney and testis. 3-MCPD esters are efficiently hydrolyzed in the gastrointestinal tract, suggesting that their toxicity is mediated by free 3-MCPD. Combined exposure to free 3-MCPD and 3-MCPD released from 3-MCPD esters might lead to dietary consumption above the tolerable daily intake of 2 µg/kg body weight/day. Suspected mechanisms of 3-MCPD toxicity include the inhibition of glycolysis and oxidative stress. Here, a comparative proteomic approach was followed to analyze the effects of 3-MCPD or 3-MCPD dipalmitate in livers from rats exposed to 10 mg/kg body weight 3-MCPD, an equimolar dose of 3-MCPD dipalmitate, or a 4-fold lower dose of the ester during a 28-day repeated-dose feeding study. Early cellular changes were monitored in the absence of overt toxicity. A comprehensive view of 3-MCPD- or 3-MCPD dipalmitate-triggered proteomic changes in rat liver links to previously proposed mechanisms of toxicity and substantially extends our knowledge on molecular hepatic effects of 3-MCPD. Organ-independent marker proteins altered upon 3-MCPD exposure, for example DJ-1/PARK7, were identified by comparison of the proteomic patterns of kidney, testis and liver.

Nonalcoholic fatty liver disease induced by 13-week oral administration of 1,3-dichloro-2-propanol in C57BL/6J mice.

1,3-Dichloro-2-propanol (1,3-DCP) is a food born chloropropanol contaminant that has been detected during the production process of a wide range of foods. In this study, we investigated the effect of 1,3-DCP on lipid metabolism of mice after 13-week subchronic exposure. The data showed that 1,3-DCP (0.05-0.5mg/kg/day) could induce nonalcoholic fatty liver disease (NAFLD) in C57BL/6J mice and the NOAEL was 0.01mg/kg/day. In addition, we studied the signaling pathway to see how 1,3-DCP worked. The data showed that NAFLD induced by 1,3-DCP was due to the dysregulation of AMPK signaling pathway. As far as we are aware, this is the first study to use 13-week subchronic toxicology to investigate the effect of 1,3-DCP on the development of NAFLD in mice. Our study provided evidence for diet contaminants in the development of NAFLD and furthered the safety evaluation of 1,3-DCP through subchronic exposure.

A 4-week study of four 3-monochloropropane-1,2-diol diesters on lipid metabolism in C57BL/6J mice.

3-Monochloropropane-1,2-diol (3-MCPD) esters have been detected in many foods, which have become a new safety issue worldwide. In the study, we investigated the effect of four 3-MCPD diesters (palmitate diester: CDP; stearate diester: CDS; oleate diester: CDO; linoleate diester: CDL) on lipid metabolism in C57BL/6J mice. The results showed that CDP, CDS, CDO and CDL significantly increased the serum TC, LDL-C levels and liver TG, TC levels at dose of 16.5µmol/kg/day. These results indicated that 3-MCPD diesters could potentially cause hyperlipidemia in C57BL/6J mice. Moreover, oil red O staining confirmed fat accumulation in liver induced by 3-MCPD diesters. Our work will provide more information for safety evaluation of 3-MCPD diesters. However, whether free 3-MCPD or free fatty acids or combined action compensates for the hyperlipidemia effects should be elucidated in the future.

Proteomic analysis of 3-MCPD and 3-MCPD dipalmitate toxicity in rat testis.

Thermal treatment of foodstuff containing fats and salt promotes the formation of 3-chloropropane-1,2-diol (3-MCPD) and its fatty acid esters. 3-MCPD-exposed rats develop testicular lesions and Leydig cell tumors. 3-MCPD and 3-MCPD ester toxicity is thought to be caused by 3-MCPD and its metabolites, since 3-MCPD esters are hydrolyzed in the gut. Inhibition of glycolysis is one of the few known molecular mechanisms of 3-MCPD toxicity. To obtain deeper insight into this process, a comparative proteomic approach was chosen, based on a 28-days repeated-dose feeding study with male Wistar rats. Animals received equimolar doses of 3-MCPD or 3-MCPD dipalmitate. A lower dose of 3-MCPD dipalmitate was also administered. Absence of histopathological changes supported an analysis of early cellular disturbance. Testes were analyzed by two-dimensional gel electrophoresis followed by mass-spectrometric protein identification. Data provide a comprehensive overview of proteomic changes induced by 3-MCPD and 3-MCPD dipalmitate in rat testis in an early phase of organ impairment. Results are compatible with known 3-MCPD effects on reproductive function, substantially extend our knowledge about cellular responses to 3-MCPD and support the hypothesis that toxicity of 3-MCPD and 3-MCPD esters is mediated via common effectors. DJ-1 was identified as a candidate marker for 3-MCPD exposure.

Effects of alpha-chlorohydrin on the testes and epididymides of dog: a preliminary study.

Chronic administration of alpha-chlorohydrin (8 mg/kg for 30 days, caused lesions in the testis of dog. Seminiferous tubules presented marked degenerative changes. Leydig cell hypertrophy was conspicous. Epididymal epithelium was regressed and the lumen was devoid of spermatozoa. Obstruction of the epididymal lumen was not seen. Alpha-Chlorohydrin inhibited the synthesis of RNA and sialic acid in the testis, caput epidiymis, corpus epididymis, and cauda epididymis. The total cholesterol per gram of testis was increased significantly after alpha-chlorohydrin administration. The anti-androgenic nature of alpha-chlorohydrin is suggested.

PIP: A preliminary study of the effects of alpha-chlorohydrin on the reproductive tract of male dogs is presented. 10 animals were injected with 8 mg/kg/day of the drug for 30 days. The seminiferous tubules showed considerable degeneration and shrinkage. A gradual loss of type A spermatogonia, spermatocytes, spermatids, and spermatozoa was observed in the testes. The volume of Leydig cell cytoplasm increased, showing a granular appearance, and the nuclei were enlarged. Epididymides weights were significantly lower than those of controls (p less than .02). The epididymal lumen contained no spermatozoa and there was no evidence of obstruction. The synthesis of RNA and sialic acid was inhibited in the testis, caput epididymis, corpus epididymis, and cauda epididymis. Alpha-chlorohydrin significantly increased the total amount of cholesterol per gram/testis (p less than .02). Results indicate that alpha-chlorohydrin is antiandrogenic in nature and acts directly on the testis and epididymal biochemistry.

Sperm motion predicts fertility in male hamsters treated with alpha-chlorohydrin.

An understanding of the relationship between altered sperm motion and sperm function (fertility) is important when interpreting the biological significance of toxicant-induced changes in sperm velocity in rodent test species. Previous studies showed that a brief (4-day) exposure of male hamsters to the model chemical alpha-chlorohydrin (ACH) results in significant deficits in epididymal and uterine sperm velocity, which are associated with both a delay and a failure of fertilization in vivo. To characterize this effect in terms of fertility, similarly treated male hamsters were bred to untreated females and pups were counted the day before parturition. ACH treatment resulted in a dose-dependent decline in the percentage of sperm-positive females that were pregnant at the end of gestation (100, 78, 67, 22, and 0 where males were treated with 0, 33, 49, 66, and 83 mg ACH/kg/day, respectively). Cauda epididymal sperm from the same males were assayed for motion characteristics using computer-assisted sperm analysis (CASA), and for fertilizing ability in vitro. While the percentage of motile sperm was unaffected by ACH treatment, sperm velocity declined in a dose-dependent manner at all ACH treatment levels. Furthermore, the velocity of sperm from infertile males was shifted downward consistently across the entire velocity distribution. Since treated males tended to either be infertile (no pups) or have near normal litter size, the correlation between sperm velocity and litter size was nonlinear. Therefore, logistic regression models using velocity cut-off values were the most useful models for predicting fertility. These results support the contention that fertility relies on there being a sufficient number of sperm that exceed a velocity threshold. Sperm from treated males were also less likely to support in vitro fertilization (IVF), providing further evidence of impaired sperm function associated with acute exposure to ACH.

Utilization of a short-term male reproductive toxicity study design to examine effects of alpha-chlorohydrin (3-chloro-1,2-propanediol).

alpha-Chlorohydrin (ACH) was administered to rats in a short-term male reproductive toxicity study to examine the usefulness of the method and to provide reference data with a substance that is known to elicit adverse effects on both sperm production and sperm quality within or following a 2-week treatment period. Adult male CD rats (10 per group) were administered ACH orally by gavage at doses of 0, 1, 5, or 25 mg/kg/day for 14 days. Males were killed on Test Day (TD) 15 or 29. A 2-week period without treatment was included to distinguish between testicular and posttesticular effects. At each time point, the reproductive system was evaluated by comparing testes weight, DNA ploidy distributions of testicular cell suspensions, testicular and epididymal histopathology, and epididymal sperm concentration, motility, morphology, and breakage. Beginning on TD 14, males to be killed on TD 29 were cohabited with untreated females (1:2). Females were killed on Gestation Day 13 and examined for pregnancy status. During the treatment period, minor depressions in body weight and relative food consumption occurred in rats administered 25 mg/kg ACH. Testicular and epididymal lesions also occurred at this dose level. DNA ploidy distributions determined by flow cytometry were predictive of testicular damage, with effects more pronounced on TD 29 than on TD 15. The preparation methods used selected for the most motile and vigorous population of epididymal sperm. Sperm motion was altered at the 5- and 25-mg/kg dose levels on TD 15. The percentage of motile sperm and the percentage of progressively motile sperm were markedly depressed at both the 5- and 25-mg/kg dose levels where antifertility effects occurred. Sperm velocities and amplitude of lateral head displacement were depressed at the 25-mg/kg dose level on both TD 15 and 29. Additionally, decreased epididymal sperm concentrations and increased breakage were recorded at this dose level. The findings in this study are consistent with the scientific literature for ACH and demonstrate posttesticular effects on epididymal sperm and delayed expression of testicular lesions. They also support the use of this methodology for an initial assessment of male reproductive effects.

An in vivo and in vitro investigation into the effects of alpha- chlorohydrin on sperm motility and correlation with fertility in the Han Wistar rat.

Following a recommendation from the International Conference on Harmonisation, pharmaceutical companies are now monitoring possible drug effects on sperm motility in the rat during preclinical safety studies by assessing sperm motility (velocity). However, it is not known precisely how changes in sperm motility relate to fertility. Therefore, the effects of alpha-chlorohydrin on sperm motility were investigated and related to fertility both in vivo and in vitro. alpha-Chlorohydrin was given orally to male rats using a range of doses: 0, 5, 10, and 20 mg/kg for at least 5 consecutive days. Sperm were than assessed for motility using a standard scoring system (operators' observation of sperm) that graded degree of motility (i.e., 0 = i mmotile to 4 = very motile). The results showed a dose-related decrease in sperm motility. The sperm also appeared to move with a "jerky" action. Surprisingly, when this was correlated to fertility, none of the females mated with treated males became pregnant. A dose-related decrease in pregnancy would perhaps have been expected. There was no effect on sperm morphology, and testicular and epididymal pathology were only seen after doses of 20 mg/kg. When sperm from untreated rats were incubated with alpha-chlorohydrin in vitro at concentrations of 0, 0.5, 1.0, and 1.5 mM, sperm motility and motion were similarly affected as observed in vivo. However, the fertilization capacity (in vitro fertilization) of the treated sperm showed a concentration-related reduction in percentage fertilization, and there was also evidence of abnormal embryo development. These findings suggest that the present standard scoring system used in preclinical safety studies is not a comprehensive indicator of sperm function and/or fertility. A better understanding of sperm movement, therefore, is desirable so effects on sperm motility can be related to fertility.

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.