Administration of capsaicin (8-methyl-N-vanillyl-6-nonenamide) to neonatal rats gives a long-lasting insensitivity to chemical irritants, and its potential as a specific toxin for peripheral C-fibers has made it of particular interest to neurobiologists concerned with pain mechanisms. The existance of capsaicin receptor on primary afferent sensory neurons is now evident. To deduce a receptor mode l for capsaicin, and propose the possible molecular interactions at the site of action, we prepared more than 50 capsaicin congeners (capsaicinoids). With these capsaicinoids, we investigated the role of functional groups in producing the long-lasting analgesia by phenylquinone writhing test and Randall-Selitto's method with ICR mice and SD rats. The structure-activity relationship of capsaicin in producing analgesia was established as follows: proper length of hydrophobic alkyl chain is 8-18 carbon atoms; 3-methoxy group of aromatic ring plays an important role but not essential; the presence of phenolic-OH is indispensable and the most suitable site is para-position; acyl amide linkage is dispensable; the linkage of amide bond bridged to the ring with CH2 is appropriate. Depletion of substance P from spinal cord and dordal horn of rats by capsaicinoids was proved by RIA and imunohistochemistry. We succeeded in eliminating a potent acute toxicity shown by capsaicin through its structural modification.
Developmental stage-dependent susceptibility to teratogens and sensitive period can be regarded as one of the essential points which characterize teratogenesis. In this paper, emphasis is given to a review of our studies on the critical developmental stage for induction of histogenetic disorders of the cerebral cortex and on the high vulnerability of developing neural cells to teratogenic agents. The undifferentiated ventricular cells in the mammalian fetal telencephalon are particularly vulnerable to various cytotoxic agents at the time when the ventricular cells start to differentiate into cerebral cortical neurons and the cortical plate appears. This period is day 13 of gestation in mice, day 15 in rats and 8 weeks after fertilization in humans. This developmental stage of the brain corresponds to the period of highest sensitivity for induction of cerebral cortical disorders observed postnatally both in laboratory animals and in humans. One of the significant factors determining the period of highest sensitivity of the ventricular cells could be a certain initial phase of cytodifferentiation in G1 and Go phase cells Prior to their acutual morphological differentiation.
Pregnant Wistar rats were given daily subcutaneous administrations of methamphetamine (MAPT; varying doses ranging from 1.0 to 4.5 mg/kg) from days 7 to 20 of gestation and teratogenic effects have been determined. The teratogenic effects inducible with orally administered caffeine (90 mg/kg/day) for the same durations were used as the positive controls. MAPT doses greater than 2.0 mg/kg have suppressed the rate of maternal Weight gain. Some of the offsprings (F1) of the prenatal MAPT treated groups had decreased growth rate and delayed development of physical characters and functional reflexes. The male offsprings of the MAPT treated groups had significant decreases in their spontaneous motor activity but had enhanced conditioned avoidance responses. However, the mating performances of these offsprings were not affected. These results indicated that prenatal exposure of MAPT may induce some behavioral teratogenicity in rats.
Various long-term bioassay methods have been used to determine the carcinogenicity of chemical substances. Among them, a long-term method developed by Toth (1968), which scores the incidence of pulmonary adenoma formation at 28 to 56 weeks following subcutaneous administration of chemical carcinogens to new-born mice is used widely. This particular long-term bioassay method, while it takes long periods, has proved useful in determining the anticarcinogenic effect of ginseng extracts against the pulmonary adenoma formation inducible with dimethylbenzanthracene, urethane, and aflatoxin B1. More recently, in order to shorten the assay durations to 9 weeks (medium-term), we have modified the existing method by adjusting the doses of carcinogens to be administered to the new-born mice. We have established a modified method in which 40-50% of mice were found to develop pulmonary adenoma 9 weeks after a subcutaneous injection of 0.5 mg/kg to new-born mice of NIH(GP) strain and this modified medium-term bioassay system was found useful in the screening of cancer preventive agents among natural products, such as ginseng and caffeine.
The purpose of this study is to evaluate the risk of carcinogenic tryptophan pyrolysis products to human health. During the last decade, new series of heterocyclic amines has been isolated as potent mutagens and later shown to be potent carcinogens in experimental animals. Among them, 3-amino-1, 4-dimethyl-5H-pyrido[4, 3-b] indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido [4, 3-b] indole (Trp-P-2), carcinogenic tryptophan pyrolysis products, have been investigated from various points of view and useful pieces of information about them have been collected. These carcinogens are widely distributed in the environment such as airborne particles, rain water, cigarette smoke and cooked foods, and they possess various pharmaco-toxicological activities such as covulsant activities and potent inhibitory effects on platelet function and dopamine metabolism. Recent investigations revealed that these compounds are present in human samples such as plasma, urine and bile, indicating that humans are actually exposed to these compound. It is a matter of urgency to establish a suitable method for monitoring the exposure levels of these compounds to humans.
Saccharomyces cerevisiae express RAD4 gene for nucleotide excision repair of UV-induced DNA damages. Upon complementation with rad<4-4> mutant, a 7.6kb clone containing the RAD4 gene designated as pPCl was isolated from a yeast genomic library. The pPCl was further narrowed to 2.5kb flanked with BglII and BartHI sites. The cloned RAD4 gene was found to propagate in E. coli without loss of its complementing activity. Pulse-field gel electrophoresis indicated that the cloned RAD4 gene was localized in the right arm of chromosome V. DNA-tRNA hybridization revealed that the cloned gene did not contain a suppressor tRNA gene. The rad4 mutants with various plasmids containing the cloned RAD4 gene, regardless of their copy number, had enhanced resistance against UV damages equivalent to that found in wild type. As determined by Sl nuclease digestion, the RAD4 transcript was found to be 2.3kb in size and the Sl nuclease mapping revealed the production of a protected fragment of 760 nucleotides within the transcript. Transcriptional start point was found at 48 base pairs upstream from the first ATG codon of the translation initiation codon. The overexpressed Rad4 protein was estimated to be 89kD and confirmed the expected size based on the actual length of RAD4 gene. Upon stationary phase culturing, E. coli cells transformed with the cloned RAD4 gene had a delayed enterance into exponential growth phase and produced reduced amount of host proteins. These results have indicated that the pPCl is a functional RAD4 gene playing a unique role involved in the nucleotide excision repair of yeast without any genetic change during amplication in E. coli.
Two in vitro tests with different genetic end points, gene mutation and chromosomal damage, and an in vivo test, preferably the micronucleus test in mice, were recommended as a battery system for the primary assessment of genotoxic effects of chemicals. From our comparative studies on the mutagenic potency of chemicals, it was pointed out that results should be evaluated quantitatively rather than qualitatively, since the potency varies extensively, at range of 1O fold, among different chemicals, and the in vitro genotoxins relatively weak tend to be negative in in vivo mutagenicity tests as well as in carcinogenicity tests in rodents. New Salmonella tester strains, called YG-series, were established, which showed a high nitroreductase or acetyltransferase activity and specifically sensitive to nitroarens or aromatic anmines in the reverse mutation assays (Ames test). These strains could detect a small amount of mutagenic aromatic amines containing in the urine of cigarette smokers. A new technique in the micronucleus test using peripheral blood erythrocytes was introduced. A cumurative genotoxic effect of benzene, for an example, was detected in the peripheral blood even several weeks after treatment every week by gavage. A cyto-flowmetric analysis can be also applied to monitoring of such effects.
Oxygen radicals have been suggested to be involved in mutation/carcinogenesis. The C-8 position of guanine residues in DNA is hydroxylated to produce 8-hydroxyguanine(8-OH-Gua) in DNA in vitro by various oxygen radical producing agents. The formation of 8-OH-Gua was also observed in cellular DNA in vivo by radiation or oxygen radical forming carcinogens. The 8-OH-Gua residue in DNA is often misread in the position of 8-OH-Gua residue itself but also at neighboring residues next to 8-OH-Gua. When second guanine in codon 12 was specifically replaced with 8-OH-Gua and transferred to NIH3T3, the recipient cells were transformed to malignant cell type. E. coli was found to contain an endonuclease which specifically recognizes 8-OH-Gua residue and cleave DNA strand before and after the modified base. The data obtained imply that 8-OH-Gua formed in DNA in vivo is recognized as an abnormal modified base which, if not repaired, play a role in the mediation of oxygen radical-involved mutation/carcinogenesis.
Among mammalian species, humans and monkeys are sole species which possess cytochrome P-450 in the liver in fetal life. Cytochrome P-450 catalyzes oxidations of a wide variety of compounds including steroids, drugs and toxicants. Thus, the purpose of this study was to examine the toxicological significance of human fetal cytochrome P-450. Firstly, we found that human fetal livers were capable of activating numbers of carcinogenic compounds. Secondly, the capacity of human fetal cytochrome P-450 to activate mutagens and carcinogens was confirmed using purified forms of cytochrome P-450. For this, we purified four forms of cytochrome P-450 from human fetal livers. Lastly, we could isolate a cDNA clone coding for P-450HFLa which is one of the major forms of cytochrome P-450 in human fetal livers. Utilizing synthesized cDNAs as probes, we found that this form of cytochrome was expressed specifically in fetuses.
Effect of administering butylated hydroxyanisole (BHA) on the metabolism of aflatoxin B1 (AFB1) and production of mutagenic metabolites have been compared with those of phenobarbital(PB) and 3-methylcholanthrene(MC) administration in rat liver microsomes. Male Sprague-Dawley rats were treated with these inducers and liver microsomes were isolated. These microsomes were used to metabolize AFB1 and to produce mutagenic metabolites. Results showed that normal rat, liver were able to metabolize AFB1 quite actively and produced large amounts of AFB-8, 9-epoxide (aprearing as the AFB-8, 9-dihydrodiol-Tris complex). Upon incubations of normal rat liver microsomes with increasing concentrations of AFB1, a steep dose-related increases of mutagenicity was observed in the Ames test. The PB-microsomes had an increased ability to metabolize AFB1 and particularly the rate for the production of the weakly mutagenic AFQ1 metabolite was markedly increased. Conversely, PB-microsomes had a moderate decrease in its ability to form the strongly mutagenic of AFB-8, 9-epoxide metabolite. However, the ability of PB-microsomes to form mutagenic metabolites from AFB-1 was somewhat greater than that of the control-microsomes. The MC-microsomes had an increased ability to metabolize AFB1 also. However instead of the weakly mutagenic AFQ1 metabolite seen with the PB-microsomes, large amounts of the strongly mutagenic AFM1 metabolite was formed. Although AFM1 is not known to be a direct mutagen, it was highly mutagenic upon activation with microsomes. The very steep dose-related increases of mutagenicity and appearance of bacterial toxicity at relatively lower doses of AFB1 may have ken caused by the secondary metabolic activation. The ability of BHA-microsomes to metabolize AFB1 was decreased. Among the metabolites produced by the BHA-microsomes, the non-mutagenic AFB<2a> was formed in significantly increased amounts but the toxic AFB-8, 9-epoxide vas produced only in much reduced amounts. The AFB<2a> was not mutagenic even after metabolic activation with microsomes. When increasing concentrations of AFB1 was incubated with BHA-microsomes, a very mild dose-related increases of mutagenicity was observed and the occurance of toxic effects on bacterial growth appeared only at high doses of AFB1. This may have ken due both to the reduced rate of overall AFB1 metabolism and to the decreased formation of the highly mutagenic AFB-8, 9-epoxide but an increased formation of the non-mutagenic AFB<2a> metabolite by the BHA-microsomes. Such a reduced rate of formation of the toxic AFB-8, 9-epoxide but an increased production of the non-toxic AFB<2a> metabolite by the BHA-microsomes may have been, at least partially. responsible for the anticarcinogenic effect of BHA.
Liver microsomal β-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein, named egasyn. In this study, we showed that egasyn is identical to one of the carboxylesterase isozymes and organophosphorus and carbamate insecticides, acetanilide which is a specific substrate of egasyn and halothane caused a rapid dissociation of the egasyn-microsomal β-glucuronidase complex when administered in vivo or when added in vitro to isolated hepatocytes. The dissociation was relatively specific to organophosphates, carbamates, but not pyrethroids. Dissociation of the egasyn-β-glucuronidase complex in vivo by organophosphates was followed by massive and rapid secretion of microsomal β-glucuronidase into plasma. From these results, we concluded that release of liver microsomal β-glucuronidase is the most rapid and sensitive marker to organophosphorus or carbamate insecticide-induced intoxication.