Molecular toxicology最新文献

The FRAME in vitro cytotoxicity assay and a physicochemical parameter for metal ions (i.e., "softness," sigma p) were assessed for their ability to predict the in vivo acute toxicities of 52 metallic compounds. The in vitro assay was found to be more useful, since it measures the toxicity of the whole compound, as does the in vivo method. The softness parameter applies to the metal ion only, so it cannot be used to predict the toxicity of compounds containing relatively nontoxic metal ions and toxic anions (e.g., potassium fluoride). The in vitro toxicity values (expressed as ID50 values, i.e., concentrations of test chemicals that reduced the final cellular protein content of test cultures by 50% in comparison with appropriate solvent control cultures) correlated better with mouse ip LD50 values than with rat oral LD50 values.

The activities of smooth endoplasmic reticulum (SER) and cytosolic xenobiotic-metabolizing enzymes were studied in two established renal tubular cell lines, LLC-PK1 and RK-L (rat kidney, Lübeck). The glutathione content in both cell lines was about 20-fold higher than in rat kidney homogenates; this is explained by a 20- to 50-fold lower activity of gamma-glutamyl transpeptidase in the cell lines. Among SER enzymes, the cytochrome P-450-dependent dimethylhydrazine demethylase was in the same range in both cell lines as compared with rat kidney S9 fraction. Pretreatment with phenobarbital (0.1 mM in the culture medium for 3 d) did not induce SER or cytosolic enzyme activities. The glutathione content in both epithelial cell lines can be modified by an inhibitor of GSH synthesis (buthionine sulfoximine, BSO), whereas inhibition of gamma-glutamyl transpeptidase (acivicin) did not significantly increase the GSH-concentration. Despite these biochemical characteristics, the utility of the new RK-L-line needs to be evaluated in experimental studies on renal transport processes and metabolism as well as cytotoxicity and genotoxicity of xenobiotics.

Oxidative damage produced by oxygen free radicals has been investigated in various mammalian cells in culture. Incubation of these cells with redox cycling quinones resulted in a stimulation of superoxide anion and hydrogen peroxide formation. Further metabolism of H2O2 by glutathione peroxidase caused oxidation and depletion of cellular glutathione followed by oxidation of protein sulfhydryl groups and cytotoxicity. Several targets susceptible to oxidative modification have been identified, including the mitochondrial, endoplasmic reticular, and plasma membrane Ca2+-translocases. As result, a marked and sustained increase in cytosolic free Ca2+ concentration occurred, followed by the activation of some catabolic Ca2+-dependent processes, namely phospholipases, proteases, and endonucleases. In addition, an impairment of the transmembranal signal-transducing system(s) was found. Recent investigations demonstrated that several modifications occur also in the cytoskeleton of oxidative stress-challenged cells. They mainly consist of oxidative actin cross-linking and dissociation of the cytoskeleton from the plasma membrane. All these alterations appear to contribute to the multifactorial process underlying the irreversible cell injury caused by oxidative stress.

Human and animal cell cultures were evaluated for their susceptibility to two environmental toxins found as contaminants in human food supplies: aflatoxin B1, a hepatotoxin produced by the mold Aspergillus flavus, and saxitoxin, a paralytic neurotoxin produced by the marine dinoflagellate Gonyaulax catenella. Both toxins cause food poisoning in humans and other animals. The acute cytotoxicity of both toxins was measured and compared by inhibition of cell growth and by progressive cytopathogenicity resulting in cell destruction. Aflatoxin B1 was cytotoxic to all of the 11 primary kidney cultures derived from susceptible animals. The cell growth inhibition 10% values (TD10) ranged from 0.02 to 6.0 micrograms/ml: mouse (TD10 = 0.02 micrograms), guinea pig (0.03 micrograms), rat (0.07 micrograms), hamster (0.16 micrograms), monkey (0.1 microgram), human (0.7-1.5 micrograms), chick (0.05 micrograms), and duck (6.0 micrograms). The corresponding TD50 levels were about 10 times higher concentrations and caused cell destruction within 2 d. Saxitoxin did not induce cytotoxicity manifestations in cultures derived from susceptible species--mouse kidney, human carcinoma HeLa line, chick embryo, and goldfish fin (CAR) cell line--at high concentration levels up to 5 micrograms/ml. When the same toxin preparation at only 1 microgram was injected into mice, the animals died immediately. The results indicate that animal cell cultures are useful for studies of general cytotoxins that affect common essential metabolism but cannot be used to detect environmental toxins that cause toxic manifestations by an interference with specific physiological functions of organ systems.

Previously, we have shown that Chinese hamster ovary (CHO) cells are useful for quantifying chemical-induced gene mutations. We have defined the conditions of a Multiplex CHO System which permits determination of mutagen-induced chromosome aberration, and sister chromatid exchange (SCE) in addition to cytotoxicity and gene mutation in the same treated culture. This allows us to extend the spectrum of quantitative mutagenesis to include clastogenic endpoints. In the present study, we used four carcinogenic/noncarcinogenic pairs to validate the relative utility and sensitivity of each endpoint, and to study the interrelationship of these four distinct biological effects. These compounds include the direct-acting carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ICR 170 and their noncarcinogenic analogue N-methyl-N'-nitroguanidine (MNG) and ICR 170-OH, and the procarcinogens benzo[a]pyrene (B[a]P) and dimethylnitrosamine (DMN) and their noncarcinogenic analogues pyrene and dimethylamine (DMA) respectively. A rat liver homogenate preparation (S9) was used to assay for the biological activities of procarcinogens. Under our experimental conditions, we observed that carcinogens DMN, B[a]P, MNNG and ICR 170, but not their noncarcinogenic counterparts, showed all four biological effects. Our studies with these chemicals showed that cytotoxicity does not necessarily correlate with any of the genetic endpoints. On a molar basis, noncarcinogens, pyrene and ICR 170-OH show similar toxicity to carcinogens B[a]P and ICR 170, respectively. The other two non-carcinogenic analogues, DMA and MNG, exhibit minimal toxicity at concentrations 10-1,000 times higher than cytotoxic concentrations of the corresponding carcinogens, DMN and MNNG. In general, gene mutation and SCE are more sensitive than chromosome aberration assay. The gene mutation assay is more specific than SCE and chromosome aberration assays since none of the noncarcinogens exhibit a detectable response in the gene mutational assay. ICR 170 and MNNG are much more active than B[a]P and DMN as ranked on a molar basis. These results indicate that the Multiplex CHO System is capable of discriminating divergent structural classes of carcinogenic and noncarcinogenic compounds, such as the eight chemicals chosen for our study.

The possibility that glutathione (GSH) S-transferases may affect microsome-mediated methylation of DNA by dimethylnitrosamine (DMN) in vitro has been investigated using aflatoxin B1 (AFB1) as a positive control. Hamster liver microsomes were incubated with either [14C]DMN or [3H]AFB1 and calf thymus DNA, with or without GSH and hamster cytosol. Although a significant amount of DMN was metabolized, GSH alone or in conjunction with cytosol or purified GSH S-transferases did not affect the binding of 14C to DNA and the amount of 7-methylguanine formed. However with AFB1, a significant reduction in both its binding to DNA and in the formation of AFB1-N7Gua adduct with a concomitant increase in AFB1-GSH conjugation was observed, suggesting that the test system was functioning effectively.

A simple, rapid, inexpensive test for teratogens has been developed using vaccinia virus growth in primate cell cultures. Eighty-four percent of the test compounds that are known to produce teratogenesis in laboratory animals, prevented the formation of viable virus at dosages that did not cause any observable cytotoxicity to uninfected cells. The virus test had one false positive and 5 false negatives out of 74 test compounds. Moreover, the 50% inhibitory dose in vitro (RD50) was significantly correlated (p less than 0.001) with the in vivo, lowest reported teratogenic dose (LTD). The RD50 was not correlated with the in vivo lethal dose (LD50). Thus the virus test appears to be more sensitive to development than to general toxicity. A comparison of the in vitro RD50 with the in vivo, rodent LTD indicated that the two tests were equally predictive of human teratogenesis.

The major defect of in vivo assays for mutagenic carcinogens may be tissue specificity: a cancer bioassay of a single tissue would not be expected to detect all carcinogens, so the failure of a genetic assay in a single tissue to detect all carcinogens should not be surprising. In the search for an environmental carcinogen responsible for a specific cancer in a particular population, however, it may be that tissue specificity can be advantageous. Assays for genotoxicity directly in the target cells may have higher success rates with fewer false positives than assays in tissues of convenience. For example, to facilitate the search for one or more dietary carcinogens responsible for the high rate of colon cancer in North America, assays for genotoxicity in the target cells themselves, the colonic epithelium, may be useful. To this end we have investigated assays for three different endpoints: nuclear anomalies, sister chromatid exchanges, and gene mutations. Our experience may prove useful for others considering a similar strategy.

The main theme of this paper is to describe the basic requirements for assembling reliable batteries of short-term tests for carcinogenicity prediction. For this purpose, a subset of the data base generated by the International Program for Evaluation of Short-Term Tests for Carcinogens (IPESTTC) has been studied by different data-analysis methods. Much attention has been focused on the methodological dimensions of the problem, at the level of selection of both data and statistical techniques. Twenty-one of the most widely used short-term assays were considered. An exploratory study of the data base was first performed by factor analysis, showing similarities and dissimilarities between test performances and confirming our previous results obtained by cluster analysis. In this way the assays were divided into three groups on the basis of their responses to the chemicals. The Salmonella assay was in the central group, characterized by equilibrated performances in respect to sensitivity and specificity for carcinogens. Tests complementary to the Salmonella assay for sensitivity and specificity, respectively, were identified as well. A preliminary comparison of the IPESTTC results with the Gene-Tox data base is also presented. The test performances in respect to carcinogenicity prediction were then evaluated by discriminant analysis. When the subset of data was considered as a whole, the procedure resulted in a linear discriminant function able to correctly identify 84.2% of carcinogens and 83.3% of noncarcinogens. The correctly identified carcinogens summed to about 90% when adequate batteries of tests were used. This analysis yielded a number of observations. (1) Together with the selectivity indices (such as sensitivity and specificity), the operational complementarity between test performances must be ascertained and taken into account. (2) The batteries most effective at predicting carcinogenic activity were composed of three tests, one for each group. This finding converged with the fact that the three classes of assays were clearly differentiated for sensitivity and specificity, and in this sense were complementary to each other. (3) The performances of the batteries were not improved by adding more tests, but in several cases the opposite effect occurred. (4) Estimation of the probability of the chemicals of being carcinogenic, starting from qualitative genotoxicity data, is possible.