Molecular toxicology最新文献

The toxicity of selected organochlorine, organophosphate, and carbamate pesticides on the functions and cellular parameters of peritoneal macrophages was examined in inbred C57Bl/6 mice. Effects of single, sublethal pesticide exposure on macrophages were determined by analysis of the cell viability, cell adherence capacity, generation of superoxide anion (O2-), antigen processing, phagocytosis of Salmonella typhimurium, and resistance to in vitro virus-induced cytopathic effects (cpe) after infection with mouse hepatitis virus 3 (MHV3). Most of the studies were done for the organochlorine pesticide dieldrin, which inhibited several macrophage functions, such as phagocytosis of S. typhimurium, release of the single processed protein antigen avidin, and resistance to MHV3 virus-induced cpe. The virus-induced cytolysis in macrophage cultures was significantly increased after in vivo exposure to single sublethal doses of other selected pesticides, such as guthion, carbofuran, sevin, and matacil. However, none of the selected pesticides, used in sublethal (0.4 less than or equal to LD50 less than or equal to 0.6) doses appeared to be a factor impairing the O2- -generating system in chemically elicited or immunologically activated peritoneal macrophages. In conclusion, sublethal pesticide exposure can induce a significant impairement of several macrophage functions, such as phagocytosis, antigen processing, and resistance to virus-induced cytolysis. Inhibition of the O2- -generating system by sublethal pesticide exposure, however, can be excluded as a mechanism of potential suppressory action of these pesticides on antiviral and antibacterial host defence systems in which macrophages play a primary role.

The effect of ethanol consumption by male CF-1 mice on liver microsomal enzyme activities has been investigated. The total microsomal cytochrome P-450 content was increased by 38%, while cytochrome b5 was decreased by 31%, which are characteristic alterations in liver microsomes following ethanol consumption. Other alterations included a decreased NADPH cytochrome c reductase activity and increased NADPH-supported rates of N-nitrosopyrrolidine and aniline hydroxylation. While ethanol consumption did not alter the total metabolism of nicotine, the rates of N- and C-hydroxylation were differently affected. The 5'-hydroxylation of nicotine was increased by 83%, while the N'-oxidation was decreased by 31%. Changes in the microsomal metabolism of the environmental carcinogen 1-nitropyrene included a slight reduction in the overall metabolism, which can be accounted for by a reduction in the formation of one phenolic metabolite, 1-nitropyren-3-ol.

Most of the heat-stable natural cytotoxins in normal human plasma were fractionated by gel filtration into two active fractions: the alpha 2-macroglobulin (alpha 2M) and pre-alpha 2M pools. The pre-alpha 2M pool also appeared to contain most of the phospholipids in human plasma. The pre-alpha 2M pool was further purified by ultracentrifugation and was shown to contain cytotoxic lipoproteins that represented most of the cytotoxin activity in the pool. The phospholipids associated with the pre-alpha 2M pool and purified lipoproteins resisted dialysis in a buffered saline but were extractable by organic solvents. Two major phospholipids (compounds A and B) were extracted, and both exhibited cytotoxic activities. Compound A exerted dose-dependent, species-nonspecific growth-inhibitory or cytotoxic effects on tumor cells in vitro. It was a lecithin-like compound but was more potent than any of the lecithin standards tested, except for a polyunsaturated lecithin. The possibilities that certain lecithin-like compound(s) contribute significantly to the cytotoxic activity of the lipoproteins and their role in cancer resistance are discussed.

Comparative studies on the reactivity of the heterocyclic nitrogen were carried out for pyridine and its three monosubstituted derivatives 2-aminopyridine (2-AP), 3-aminopyridine (3-AP), and 4-aminopyridine (4-AP) to reveal the structural basis for the differences in their susceptibility to N-oxidation. Molecular orbital calculations were performed to obtain the wave functions for the calculation of the molecular electrostatic potentials (MEP) generated by the molecules. The comparison of the reactivity of the cyclic nitrogen, evaluated from the depth and accessibility of the minimum in the MEP, indicates that the nitrogen in 4-AP will be most susceptible to protonation and will be the most protected from N-oxidation at physiological pH values. The MEP map for 2-AP reveals the smallest minimum in the series of compounds and a considerable reduction in the accessibility of the region near the cyclic nitrogen caused by the proximal substitution. On this basis, 3-AP becomes the most likely derivative to form the ring N-oxide. Comparison of the conclusions from the MEP analysis with available data from bioassays suggests that the mechanism responsible for the genotoxic effects of the chemicals, where only 3-AP is active, is very different from the mechanism for systemic toxicity where 3-AP is the least active, and 4-AP is most active probably due to its channel blocking properties. As the mechanisms for the biological activities of the N-oxide metabolites become clear, reliable predictions of the toxicity of the pyridines should become possible based on such reactivity characteristics.

Toxic properties of the mycotoxins cyclopiazonic acid and aflatoxin B1 have been analyzed separately and in combination by monitoring their effects on luminescence in the marine bacterium Photobacterium phosphoreum, Strain NCMB 844. Genotoxicity was analyzed with a dark mutant of this organism whose reversion to the bioluminescent condition is stimulated by compounds attacking guanine sites in deoxyribonucleic acids. In this assay, cyclopiazonic acid, unlike aflatoxin B1, is not enhanced by cyclopiazonic acid when the two mycotoxins are assayed in combination. Cytotoxicity was assessed by the diminution of bioluminescence in a separate assay system with strain NRRLB-1177 of P. phosphoreum. Cyclopiazonic acid is more cytotoxic than aflatoxin B1, and concentrations of cyclopiazonic acid required for cytotoxicity decreases with time, whereas aflatoxin B1 cytotoxic expression does not change significantly with time under most assay conditions. Aflatoxin B1 and cyclopiazonic acid assayed as a dose pair indicate that these mycotoxins elicit their effects by independent modes of action.

Several mechanisms have been postulated to be responsible for the pleiotropic effects of toxic chemicals. Although the cytotoxicity and mutagenicity of chemicals are well studied and relatively easily detected, the noncytotoxic and nonmutagenic (i.e., epigenetic) mechanisms of chemical toxicity are less well understood. An in vitro assay, using cocultures of Chinese hamster cells to measure metabolic cooperation between V79 6-thioguanine-sensitive (6TGs) and resistant (6TGr) cells, has been developed to detect noncytotoxic and nonmutagenic chemicals that inhibit, quantitatively, gap junctional communication. The insecticides aldrin, dieldrin, and toxaphene, known to have pleiotropic toxic effects in animals, were shown to inhibit gap junctional communication. Interpretation of results suggests that chemical inhibition of gap junctional communication could be a possible mechanism to explain their tumor-promoting and neurotoxic effects.

Multiple reactions are thought to be involved in transforming dialkynitrosamines to active carcinogens. The first proposed step is enzymatic alpha-hydroxylation by the active oxygen species of cytochrome P-450, followed by nonenzymatic N-dealkylation and formation of diazohydroxides (RNNOH). The latter transformation can reasonably occur by a two-step mechanism via tautomerization of a monoalkylnitrosamine intermediate or directly from the alpha-hydroxylated species in one step. Both of these pathways in the transformation of hydroxymethylnitrosamine to diazohydroxide and formaldehyde were examined by the semiempirical molecular orbital method MNDO (modified neglect of diatomic differential overlap) and the ab initio method using STO-3G and 3-21G basis sets. Complete geometry optimizations of all reactants, intermediates, transition states, and products were performed. MNDO was also used to compare the similar transformation of the dimethyl analog. Both methods show that in the gas phase a concerted pathway involving a six-membered ring transition-state pathway is kinetically favored over a two-step pathway involving N-demethylation followed by tautomerization via two four-membered ring transition states. This reaction appears to be a viable one to formation of an ultimate carcinogen by parent dialkylnitrosamines in the hydrophobic substrate binding site of cytochrome P-450.

beta,beta'-Iminodipropionitrile (IDPN) induces neurobehavioral aberrations in experimental animals and massive focal accumulations of neurofilaments in proximal regions of axons. A hypothesis is presented to explain the neurotoxic activity of IDPN in terms of oxidative amine metabolism, wherein a resonance-stabilized cyanoenamine 3-(2-cyanoethylamino)acrylonitrile (dehydro-IDPN, 5) could be generated. Chemical studies were conducted to verify the likelihood of the proposed enzymatic transformations and their consistency with the known excreted metabolites. Dehydro-IDPN gives rise to a slow hydrolytic release of cyanoacetaldehyde at pH 7, which can transform protein-based amino groups to cyanoenamines, though the latter derivatives could be formed directly through a relatively rapid transamination reaction with dehydro-IDPN at pH 7. Kinetic studies were conducted to assess the balance between competing hydrolysis (pseudo-first order) and transamination (second order) of cyanoenamines as a function of pH. Cyanoethenylation of the epsilon-amino groups of critical lysine residues in the "tail-piece" domains of neurofilament (NF) subunit proteins could disrupt the supramolecular coulombic interactions thought to contribute to maintenance of cytoskeletal caliber. This could result in a defect in the slow axonal transport of NF, and subsequently in the formation of proximal axonal enlargements.

The carcinogenicity prediction and battery selection (CPBS) method can be used to predict the probable carcinogenicity of a chemical based on the results of a battery of short-term assays. The method uses Bayesian statistics and the estimated performance characteristics of the assays (i.e., sensitivity and specificity). For routine use, the prior probability of carcinogenicity (or of noncarcinogenicity) is assumed to be unknown and is assigned a nondiscriminatory value of 0.5, i.e., the chemical is assumed to have an equal probability of being a carcinogen or a noncarcinogen, which implies that the expert's intuition regarding the chemical's potential as a carcinogen, based on structural features, known metabolic transformation, or potential electrophilicity, is not taken into consideration. In the present study, it is shown with cyclamate and its metabolite cyclohexlamine that when a battery of assays is used, assigning values to the prior probability between 0.1 and 0.9 has no significant effect on the predicted carcinogenicity. In the view of the fact that the performance of short-term tests is calibrated against known carcinogens and noncarcinogens, and since in the available data bases there is a preponderance of carcinogens, it may be argued that the estimation of sensitivities may be biased toward elevated values. It is shown, however, that when a battery of assays is used, assigning decreased values to the sensitivity does not result in significant effects on the predicted activity of the two test chemicals. Frequently, in the compilation of short-term results within a single assay, different laboratories may report varying results. Heretofore the "consensus result" was derived by majority rule. Because the variability in results may have biological significance, and in view of the fact that for a widely used sweetner one might be even more risk-adverse, a modification of Bayes's formula was derived to take these differences into consideration when calculating the probable carcinogenicity of cyclamate and its major metabolite.

The carcinogenic activities in rats and hamsters and the mutagenic activity in Salmonella of a number of N-nitroso compounds belonging to various classes have been compared. While most directly acting N-nitroso compounds and those requiring metabolic activation are mutagenic with appropriate activation and seem to alkylate DNA in vivo, there are exceptions. Some of these are mutagens that are not carcinogenic; others are carcinogens that are nonmutagenic. Even among the mutagenic carcinogens, there is no quantitative relationship between mutagenic and carcinogenic activities. This implies to directly acting compounds and to those requiring metabolic activation. The lack of congruence between the two activities among the nitrosamines is due to the complexity of the metabolic activating processes leading to formation of proximate carcinogens. The deficiencies in the mutagenesis assay appear to arise from a lack of the necessary enzymes in the liver microsomal fractions used for activation. Nitrosamines bearing oxygen on the beta carbon of an alkyl chain are not oxidized by rat microsomal enzymes and hence are not converted to bacterial mutagens by rat liver microsomes. Bacterial mutagenicity is not a guide to carcinogenic activity of N-nitroso compounds or to the mechanisms by which these compounds induce cancer.