Successful validation of genomic biomarkers for human immunotoxicity in Jurkat T cells in vitro.

Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity.

HOCl-modified phosphatidylcholines induce apoptosis and redox imbalance in HUVEC-ST cells.

Electrophilic attack of hypochlorous acid on unsaturated bonds of fatty acyl chains is known to result mostly in chlorinated products that show cytotoxicity to some cell lines and were found in biological systems exposed to HOCl. This study aimed to investigate more deeply the products and the mechanism underlying cytotoxicity of phospholipid-HOCl oxidation products, synthesized by the reaction of HOCl with 1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, and 1-stearoyl-2-arachidonyl-phosphatidylcholine. Phospholipid chlorohydrins were found to be the most abundant among obtained products. HOCl-modified lipids were cytotoxic towards HUVEC-ST (endothelial cells), leading to a decrease of mitochondrial potential and an increase in the number of apoptotic cells. These effects were accompanied by an increase of the level of active caspase-3 and caspase-7, while the caspase-3/-7 inhibitor Ac-DEVD-CHO dramatically decreased the number of apoptotic cells. Phospholipid-HOCl oxidation products were shown to affect cell proliferation by a concentration-dependent cell cycle arrest in the G0/G1 phase and activating redox sensitive p38 kinase. The redox imbalance observed in HUVEC-ST cells exposed to modified phosphatidylcholines was accompanied by an increase in ROS level, and a decrease in glutathione content and antioxidant capacity of cell extracts.

Inhalation carcinogenicity of epichlorohydrin in noninbred Sprague-Dawley rats.

Inhalation exposure experiments with the direct-acting alkylating agent epichlorohydrin (ECH) were done on noninbred male Sprague-Dawley rats. Single 6-hour exposure to ECH and follow-up for 14 days showed the median lethal concentration to be about 360 ppm. Further inhalation experiments were done with 6-hour exposure 5 days/week. A short-term 30-exposure regimen with 100 ppm ECH produced malignant squamous cell carcinomas of the nasal cavity in 15 of 140 rats and respiratory tract papillomas in 3 rats. Among 100 rats, lifetime exposure to 30 ppm yielded 1 malignant squamous carcinoma of the nasal cavity plus 1 nasal papilloma. No nasal or respiratory tract tumors were produced by lifetime exposure of 100 rats to 10 ppm. As controls, 100 air-treated and 50 untreated rats were used. A dose-rate effect was observed for ECH inasmuch as 30-day exposure to 100 ppm (3,000 ppm-days) produced 15 cancers in comparison to the 1 cancer from the lifetime exposure to 30 ppm (8,700 ppm-days) and no cancers from lifetime exposure to 10 ppm (2,500 ppm-days).

Disruption of spindle checkpoint function in rats following 28 days of repeated administration of renal carcinogens.

We previously reported that 28-day exposure to hepatocarcinogens that facilitate cell proliferation specifically alters the expression of G1/S checkpoint-related genes and proteins, induces aberrant early expression of ubiquitin D (UBD) at the G2 phase, and increases apoptosis in the rat liver, indicating G1/S and spindle checkpoint dysfunction. The present study aimed to determine the time of onset of carcinogen-specific cell-cycle disruption after repeated administration of renal carcinogens for up to 28 days. Rats were orally administered the renal carcinogens nitrofurantoin (NFT), 1-amino-2,4-dibromoantraquinone (ADAQ), and 1,2,3-trichloropropane (TCP) or the non-carcinogenic renal toxicants 1-chloro-2-propanol, triamterene, and carboxin for 3, 7 or 28 days. Both immunohistochemical single-molecule analysis and real-time RT-PCR analysis revealed that carcinogen-specific expression changes were not observed after 28 days of administration. However, the renal carcinogens ADAQ and TCP specifically reduced the number of cells expressing phosphorylated-histone H3 at Ser10 in both UBD(+) cells and proliferating cells, suggestive of insufficient UBD expression at the M phase and early transition of proliferating cells from the M phase, without increasing apoptosis, after 28 days of administration. In contrast, NFT, which has marginal carcinogenic potential, did not induce such cellular responses. These results suggest that it may take 28 days to induce spindle checkpoint dysfunction by renal carcinogens; however, induction of apoptosis may not be essential. Thus, induction of spindle checkpoint dysfunction may be dependent on carcinogenic potential of carcinogen examined, and marginal carcinogens may not exert sufficient responses even after 28 days of administration.

Carcinogenicity study of trichloroethylene, with and without epoxide stabilizers, in mice.

Previous analytical studies of industrial samples of trichloroethylene (TRI) have revealed the presence of mutagenic and carcinogenic epoxides which, it was proposed, might be responsible for the carcinogenicity of such samples, as demonstrated with mice in other laboratories. To test this hypothesis, Swiss mice (ICR/HA) of both sexes, bred and kept in SPF conditions, were dosed daily with TRI in corn oil by gavage (males: 2.4 g/kg, females: 1.8 g/kg) with or without the addition of epichlorohydrin (EPC, 0.8%, w/w), 1,2-epoxybutane (BO, 0,8%), or EPC + BO (0.25% + 0,25%) for 18 months. The ensuing observation period terminated at 106 weeks (from start of experiment). Gross and microscopic examination of all organs revealed a statistically significant increase in the incidence of forestomach papillomas and carcinomas after EPC-, BO-, and (EPC + BO)-stabilized samples of TRI, but not after pure, amine base-stabilized TRI. This type of tumor is believed to be induced by the direct alkylating epoxides epichlorohydrin and epoxybutane, whose industrial use in stabilizing chlorinated aliphatic hydrocarbons should be discontinued. No other significant increase in tumor incidences was found. Again, this study does not support the suggestion that trichloroethylene itself is carcinogenic under realistic exposure conditions.

Chemical mutagenesis testing in Drosophila. IX. Results of 50 coded compounds tested for the National Toxicology Program.

Fifty chemicals were tested for mutagenic activity in post-meiotic and meiotic germ cells of male Drosophila melanogaster using the sex-linked recessive lethal (SLRL) assay. As in the previous studies in this series, feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. One gaseous chemical (1,3-butadiene) was tested only by inhalation. Those chemicals that were mutagenic in the sex-linked recessive lethal assay were further tested for the ability to induce reciprocal translocations. Eleven of the 50 chemicals tested were mutagenic in the SLRL assay. These included bis(2-chloroethyl) ether, 1,4-butanediol diglycidyl ether, 1-chloro-2-propanol, dimethyl methylphosphonate, dimethyl morpholinophosphoramidate, dimethyloldihydroxyethylene urea, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isatin-5-sulfonic acid (Na salt), isopropyl glycidyl ether, and urethane. Five of these, including 1,4-butanediol diglycidyl ether, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isopropyl glycidyl ether, and urethane, also induced reciprocal translocations.

Carcinogenicity study with epichlorohydrin (CEP) by gavage in rats.

Weanling Wistar rats of both sexes were given epichlorohydrin by gastric intubation for 2 years, 5 times a week at dosages of 0, 2, and 10 mg/kg body weight. Mortality and body weight gain were recorded and histopathological examination for tumours was carried out; after 1 year also haematology was performed. Towards the end of the study a slight dose-related increase in mortality was observed in males, along with a decrease in mean body weight in the survivors. At pathological examination a high incidence (100% for females, 81% for males) of squamous cell carcinomas of low-grade malignancy was observed in the forestomach of animals at risk (greater than 18 months) from the 10 mg/kg group. In the 2 mg/kg group forestomach tumours were found at a lower incidence (7% for females, 14% for males), whereas this tumour was not found in control animals. Other tumours diagnosed in this study occurred at background level and were not influenced by treatment.

Involvement of cytochrome P4502E1 in the toxicity of dichloropropanol to rat hepatocyte cultures.

Hepatocytes were isolated and cultured from untreated rats and rats treated with isoniazid to induce cytochrome P4502E1. Isoniazid selectively increased p-nitrophenol hydroxylase activity in 2-h cultures, and increased the toxicity of both 1,3- and 2,3-dichloropropanol. Isoniazid also increased the rate and extent of glutathione depletion by the dichloropropanols. There was no effect of isoniazid on the toxicity of 1,3-dichloroacetone, precocene II or allyl alcohol. In addition, diethyldithiocarbamate selectively inhibited p-nitrophenol hydroxylase in 2-h cultures from untreated and isoniazid-treated rats, as well as abolishing toxicity of the dichloropropanols. In 24-h cultures from isoniazid-treated rats diethyldithiocarbamate inhibited high affinity MCOD activity by 55% and there was also a small but significant inhibition of precocene II toxicity. These results indicate that isoniazid-inducible P4502E1 can mediate the toxicity of dichloropropanol.

Fatty acid chlorohydrins and bromohydrins are cytotoxic to human endothelial cells.

Reaction of unsaturated lipids with the hypohalous acids (hypochlorous acid and hypobromous acid) results in the addition of the halide (X) across double bonds to form halohydrins (-CH2CH(OH)CH(X)CH2-). These modified lipids could be potentially destabilising to cell membranes due to their increased polarity. We have investigated the effect of pre-formed halohydrins on human umbilical vein endothelial cells (HUVEC) by incubating cultured cells with oleic acid micelles containing chlorohydrins or bromohydrins. Cell detachment and necrotic death were observed with increasing doses of halohydrins, whereas the cells were unaffected by equivalent doses of oleic acid. Bromohydrins caused more lysis than did chlorohydrins at equivalent doses. Complete lysis was seen with 200 microM fatty acid/chlorohydrin micelles and with 50 microM fatty acid/bromohydrin micelles. Chlorohydrin uptake was much less than the oleic acid control whereas bromohydrins were incorporated into the endothelial cells similarly to oleic acid. This difference or the bulkier nature of the bromohydrins could account for their increased toxicity. This study has demonstrated the potential toxicity of the halohydrins, and implications for their formation in inflammation are discussed.

Effect of cyanamide on toxicity and glutathione depletion in rat hepatocyte cultures: differences between two dichloropropanol isomers.

The effect of aldehyde dehydrogenase inhibition by cyanamide pre-treatment in vitro on dichloropropanol-dependent toxicity and glutathione depletion was investigated in 24 h rat hepatocyte cultures. Cyanamide pre-treatment had no effect on nitrophenol hydroxylase, 7-methoxy-, 7-ethoxy- or 7-benzyloxyresorufin O-dealkylase activities in 24 h cultures from untreated rats, and had no effect on intracellular glutathione content in cultures from untreated rats, or in cultures from isoniazid-treated rats in which cytochrome P4502E1 (CYP2E1) is increased. In cultures from untreated animals the primary alcohol, 2,3-dichloropropanol, was not toxic and did not significantly deplete glutathione. Cyanamide pre-treatment however, potentiated both toxicity and glutathione depletion. Induction of CYP2E1 also potentiated the toxicity of 2,3-dichloropropanol, and in these cultures cyanamide pre-treatment significantly increased both toxicity and glutathione depletion. Cyanamide did not alter the toxicity or glutathione depletion due to the secondary alcohol, 1,3-dichloropropanol, irrespective of CYP2E1 induction. These results indicate that the primary alcohol isomer is metabolised to an aldehyde intermediate which depletes glutathione. Under basal conditions this metabolite appears to be effectively detoxified, but increased CYP2E1 activity and/or decreased aldehyde dehydrogenase activity promotes accumulation of metabolite, and therefore increases glutathione depletion and toxicity.

The effect of the isomers of alpha-cholorohydrin and racemic beta-chlorolactate on the rat kidney.

The (R)- and (S)-isomers of the male antifertility agent alpha-chlorohydrin have been synthesized. When administered to rats, the (R)-isomer induced a period of diuresis and glucosuria, whereas the (S)-isomer, which possesses the antifertility activity, had no detrimental action on the kidney. Neither of the isomers of alpha-chlorohydrin nor those of an active analogue, 3-amino-1-chloropropan-2-ol, had any inhibitory activity on the oxidative metabolism of glucose or lactate in isolated kidney tubules. However, beta-chlorolactate, a metabolite common to both compounds, inhibited the oxidation of glucose, lactate, pyruvate and glutamate to CO2. It is proposed that the antifertility action of the (S)-isomers of alpha-chlorohydrin and 3-amino-1-chloropropan-2-ol is unrelated to the renal toxicity of the (R)-isomers, a toxic action involving the inhibition of oxidative metabolism by (S)-beta-chlorolactate or a further product of this metabolite.

Quantitative assessment of spermatogenesis in rats given 1-amino-3 chloro-2 propanol hydrochloride (CL 88, 236).

A quantitative histomorphometric assessment of testicular spermatogenesis was undertaken on testes from rats which had received 1-amino-3 chloro-2 propanol hydrochloride (CL 88, 236) at oral doses of 0, 50, 250 or 500 mg/kg/day for 12 weeks. Rats which developed epididymal sperm granulomata or severe atrophy of the germinal epithelium were excluded from quantitative examination. Pathological changes in the epididymis and semininferous epithelium were not strongly correlated. CL 88, 236 administered at 50 mg/kg/day was without effect on the histomorphometry of the seminiferous epithelium, although epididymal lesions occurred at this dose. At higher doses a quantitative reduction in testicular spermatids was evident. It appears important to differentiate between the selective antifertility action of CL 88, 236 on the biochemistry of epididymal spermatozoa and the disruption of epididymal physiology, and testicular spermatogenesis found at unusually high doses.

Inhibition of alcohol dehydrogenase by chloral hydrate and trichloroethanol: possible role in the chloral hydrate-ethanol interaction.

Both chloral hydrate and trichloroethanol inhibited mouse liver alcohol dehydrogenase (LADH) in vitro. The inhibition of LADH by chloral hydrate appears to be non-competitive in nature with an inhibition constant (Ki) of about 2.7 X 10(-4) M. The inhibition of LADH by trichloroethanol was competitive and the (Ki) was about 2.7 X 10(-5) M. The elimination of ethanol from the blood and brain was significantly reduced in chloral hydrate- or trichloroethanol-pretreated mice. Since reduced elimination of ethanol could result in the prolongation of its central depressant activity, we suggest that this should be considered as a factor in the enhanced pharmacological effects of ethanol-chloral hydrate mixtures.

Genotoxicity, metabolism and blood kinetics of epichlorohydrin in mice.

Epichlorohydrin (ECH), a direct mutagen in vitro, did not induce chromosomal aberrations in bone-marrow cells of CD1 mice given single oral doses of 50 and 200 mg/kg in water. The ECH diol derivative (3-chloro-1,2-propanediol) was tested in vitro by a forward-mutation assay on the yeast Schizosaccharomyces pombe and showed a weak but significant mutagenic effect. The failure of ECH to induce mutagenic effects appears to be due to the rapid metabolic clearance of the compound in vivo. ECH blood kinetics at both doses, and at the same time the concentration of the diol, were determined. ECH rapidly disappeared from mouse blood, being no longer detectable 20 min after treatment. In contrast, 3-chloro-1,2-propanediol was measurable up to 5 h after dosage. No difference was observed in the kinetic and metabolic behavior of ECH after single and repeated doses (50 and 200 mg/kg/day for 7 days). When 3-chloro-1,2-propanediol was tested, neither glutathione depletion nor epoxide hydrolase inhibition (evaluated with both styrene-7,8-oxide and ECH as substrates) could be detected in mouse liver. Finally, no difference in ECH blood kinetics or metabolism were observed in experiments in which the compound was administered (200 mg/kg) intraperitoneally in water or orally as a solution in dimethyl sulfoxide.

Mutagenic relevance of rat hepatocyte nuclei in the activation and inactivation of xenobiotica. Cyclophosphamide and epichlorohydrin activity on the yeasts S. pombe and S. cerevisiae.

The influence on the mutagenicity of cyclophosphamide (Cy) and epichlorohydrin (ECH), of liver nuclei and hepatic post-mitochondrial (S9) preparations from phenobarbital-treated rats, was examined. The study was conducted in vitro, with 2 yeasts, Schizosaccharomyces pombe (P1 strain) which allows the detection of forward mutations, and Saccharomyces cerevisiae (D5 strain), in which the induction of different genetic effects, such as mitotic recombination, can be evaluated. The results indicated that the nuclear fraction has a qualitative capacity for biotransformation of compounds, overlapping that of S9. From a quantitative point of view, the Cy-activating capacity of the nuclear fraction was twice as high as that of S9 whereas the two fractions showed a similar ECH-inactivating ability. The present study strengthens the hypothesis of the relevance of nuclei as a site of metabolic activation and inactivation of exogenous compounds.

Some preliminary observations of the nephrotoxicity of the male antifertility drug (+/-)alpha-chlorohydrin.

(+/-)alpha-Chlorohydrin (80 mg kg-1) given by mouth to group-housed adult male Sprague Dawley rats produced an increase in the weight of the kidneys which persisted for at least 7 days, but there were no deaths. This dose administered to Sprague Dawley rats caged singly killed 3 out of 8 animals. The toxicity was studied in more detail using Wistar rats caged singly in metabolic cages. 4 out of 9 animals died with oliguria and anuria after 120 mg kg-1 (+/-)alpha-chorohydrin, 100 and 120 mg kg-1 (in the surviving animals), produced a loss in appetite and body weight, proteinuria, a dose-related diuresis and an increased water intake. Urinary glucose was dramatically elevated after 100 mg kg-1 but after 120 mg kg-1 the glucosuria was not as marked. By day 7 all parameters were returning to their pre-injection values. A dose of 80 mg kg-1 had no effect upon any of the parameters studied. The results are discussed in relation to the basic biochemical mechanism by which the drug exerts its antifertility action, which is achieved at much lower doses.

Mutagenic and carcinogenic risk of oxygen containing chlorinated C-3 hydrocarbons: putative secondary products of C-3 chlorohydrocarbons and chlorination of water.

Oxygen containing C-3 chlorohydrocarbons are secondary products of C-3 chlorohydrocarbons formed during oxidation at air, in the metabolism of pesticides and by chlorination of drinking water. These compounds are mutagenic, genotoxic and carcinogenic. 2-Chloroacroleins are extremely strong mutagens and genotoxins and form 1,N2-cyclic deoxyguanosine adducts. The role of such adducts in mutagenicity and carcinogenicity is discussed.

Toxic, DNA-damaging and mutagenic activity of epichlorohydrin on human cells cultured in vitro.

Epichlorohydrin (ECHH) highly inhibited the tritiated thymidine uptake by human lymphocytes cultured in vitro, although the corresponding cell viability was unaffected. Furthermore, it elicited unscheduled DNA synthesis, acting as a DNA-damaging agent after its metabolic activation. ECHH also showed a clear toxic and mutagenic activity toward a human epithelial-like cell line, causing a decrease in cell viability and an increase in mutants resistant to 0.05 Lf/ml of diphtheria toxin.

[Toxicity of dichloropropanols].

A rare outbreak of acute hepatic damage in workers exposed to dichloropropanols was reported in 1992. As there are no detailed reports of dichloropropanols (DCPs) toxicity and its mechanism, we reviewed the toxicity of dichloropropanols using our results. 1) A marked elevation of serum AST and ALT with massive necrosis of the liver was noted in the 1/2 x, the 1 x and 2 x LD50 (0.149 mg/kg) of 1, 3-dichloro-2-propanol(DC 2 P). Hepatic malondialdehyde level was significantly increased, and associated with a decrease in liver glutathione S-transferase activity and reduced glutathione content. It is suggested that the free radical is associated with DCPs. 2) A reduction of leukocytes, platelets and fibrinogen, and prolonged prothrombin time were observed in the 1 x LD50 of DC 2 P. 3) In the CA1 area of the hippocampus, inhibition of population spikes was reduced by the 1 x LD50 of DC 2 P. This research was completed with the assistance of several other papers concerning dichloropropanols toxicity.

[Toxicity of dichloropropanols--changes in hematological findings and serum chemistry].

We investigated the toxicity of dichloropropanols (DCPs) in hematological findings and serum chemistry. The solutions of two isomers of DCPs, 1,3-dichloro-2-propanol (DC2P) and 2,3-dichloro-1-propanol (DC1P) were dissolved in saline at the concentration of 100 mg/ml, and 0.1 ml of each solution was subcutaneously injected into male Wistar rats weighing about 200 g. At 6 hours after the injections, in the DC2P group, the number of white blood cells and platelets showed a significant decrease. Transaminases, alkaline phosphatase and lactate dehydrogenase were greatly elevated. Blood urea nitrogen and creatinine also showed a significant increase. There were no changes in the measurements in the DC1P group. These results indicate that there is a prominent hepatotoxicity in DC2P, and that there is a considerable difference in the toxicity present in DC2P and DC1P. Furthermore, in the workplace where DCPs, especially DC2P, is used, the monitoring of the working environment and biological monitoring should be mandatory.