The role of adrenal corticosteroids in induction of micronuclei by morphine.

It has previously been shown that morphine can increase the frequency of micronucleated splenocytes when administered to mice, but not when cells are exposed to the opiate in vitro. Morphine treatment is also known to increase circulating levels of glucocorticosteroids, which have been reported to produce genetic damage in vivo and in vitro. In order to determine whether adrenal hormones might mediate the genotoxic effects of morphine, adrenalectomized and sham-operated mice were treated with morphine sulfate. In sham-operated animals administration of morphine produced a dose-related increase in the frequency of micronucleated cells, whereas adrenalectomy abolished the effect. When plasma from morphine-treated mice was used to supplement growth medium of untreated splenocytes, the frequency of micronucleated cells increased, an effect partially blocked by the steroid antagonist RU 486. The N-methylmorphine, which does not stimulate the release of corticosterone from adrenal glands, induced micronuclei formation in splenocytes, and administration of metyrapone, an inhibitor of corticosterone biosynthesis, blocked the morphine-induced increase in corticosterone secretion, but had no effect on the frequency of micronuclei formation. These results indicate that basal levels of glucocorticosteroids are required for induction of micronuclei by morphine in murine splenocytes, but activation of the hypothalamo-pituitary-adrenal (HPA) axis by morphine does not contribute to the observed response.

Carcinogenesis: basic principles.

Many steps are required to convert a normal cell into a cancerous one. The cancer cell must be able to multiply under conditions that a normal cell would not and to invade surrounding tissue and spread throughout the body. Both genetic changes, such as activation of oncogenes or inactivation of tumor suppressor genes, and epigenetic changes, such as stimulation of cell proliferation, contribute to the development of cancers. Chemical agents can increase the probability of malignant transformation by inducing mutations that can ultimately lead to tumor formation, by promoting the development of tumors in cells with preexisting genetic damage, or by increasing the rate of acquisition of malignant traits by benign tumors. Chemical carcinogens are structurally diverse, but all initiating agents are either already electrophiles or can be converted to electrophilic reactants through metabolic activation. Genetic and environmental factors can alter an individual's ability to metabolize carcinogens, to repair DNA damage, and to respond to mitogenic stimuli, all of which can alter susceptibility to chemical carcinogenesis. The incidence and time required for appearance of tumors appear to be dose-related, but the existence of no-effect doses of carcinogens remains controversial.

Continuous Measurement of Changes in Intracellular Calcium Concentration in Mouse Splenic T Cells Attached to a Glass Substrate.

Mitogen- and isoproterenol-induced changes of [Ca(2+)](i) in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak((R)) (3.5 &mgr;g/cm(2)). The cells were then loaded with fluorescent dye (2 &mgr;g/ml of fura2-AM or fluo3-AM) and changes in [Ca(2+)](i) in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca(2+)](i) demonstrated that concanavalin A (Con A, 2 or 5 &mgr;g/ml) caused an increase in [Ca(2+)](i) that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05-5 &mgr;g/ml) had an EC(50) of approximately 0.3 &mgr;g/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca(2+)](i). In single cell studies, the increase in [Ca(2+)](i) in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together these data demonstrate that Cell Tak((R)) can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells. Copyright 1995 S. Karger AG, Basel

The clastogenicity of morphine sulfate in vivo.

An opioid analgesic, morphine, and an opioid peptide, beta-endorphin, have been shown to induce chromosome damage, as indicated by an increased frequency of micronucleated lymphocytes, following acute administration to mice. The genotoxic response is opioid receptor-mediated and is abolished in adrenalectomized animals. Further, plasma from morphine-treated animals also induces micronuclei formation in naive lymphocytes in vitro; this response is blocked by inclusion the steroid antagonist RU 486 in the incubation mixture. In addition to the steroid-mediated production of chromosome damage, morphine acts directly on lymphocytes to enhance the clastogenicity of acutely administered cyclophosphamide in manner consistent with depressed DNA repair capacity.

Induction of micronuclei in murine lymphocytes by morphine.

Although individuals who abuse drugs are prone to an increased risk of malignancy, the mutagenic and carcinogenic potential of these agents has received relatively little attention. We report here on the potential of morphine to induce micronuclei in murine lymphocytes. Following a single intraperitoneal injection of 20 mg/kg morphine, the frequency of micronucleated binuclear (cytochalasin-blocked) murine T- and B-splenocytes was elevated from 12-36 hr after treatment. The maximum frequencies seen 24 hr after injection were 6.3- and 4.9-fold greater than the respective controls. A dose-dependent induction of micronuclei was observed from 5-20 mg/kg morphine, with no further increases in frequency produced by higher doses. In contrast, incubation of mitogen-stimulated splenocytes with 10(-7)-10(-4) M morphine in vitro produced no change in frequency of micronucleated cells relative to controls. Treatment with the narcotic antagonist naloxone (5 mg/kg) alone had no effect on the frequency of micronuclei, but reduced the clastogenic response of a subsequently administered dose of morphine (20 mg/kg). Thus, in murine lymphocytes morphine indirectly produces genetic damage, which is at least in part opioid receptor-mediated.

The effect of biotransformation of 2,4-dinitrotoluene on its mutagenic potential.

Because both oxidative and reductive metabolism of the hepatocarcinogen 2,4-dinitrotoluene (2,4-DNT) can occur in vivo, we have examined the mutagenicity of compounds which can be formed from 2,4-DNT in an attempt to establish which metabolic pathways contribute to the formation of genotoxic products. A quantitative reversion assay using Salmonella typhimurium TA98 was used to evaluate the mutagenicity of these compounds. 2,4-Dinitrobenzyl alcohol, 2-amino-4-nitrotoluene and 2-nitroso-4-nitrotoluene were found to be more mutagenic to S. typhimurium than is 2,4-DNT and did not require metabolic activation by post-mitochondrial supernatants of Aroclor-induced rat liver homogenates (S9) for their effect. 2-Amino-4-nitrobenzoic acid was also mutagenic to S. typhimurium TA98 in the absence of S9, but its mutagenicity was enhanced when S9 was included in the incubation mixture. 2,4-Diaminotoluene required S9 for demonstration of mutagenicity and was approximately as effective, on a molar basis, as 2,4-DNT in inducing reversion to histidine prototrophy. These results suggest that both oxidative and reductive metabolism may be involved in production of mutagenic metabolites of 2,4-DNT.

Genotoxicity of two fecal steroids in murine colonic epithelium assessed by the sister chromatid exchange technique.

Two components of human feces are known to induce nuclear anomalies in mice when applied intrarectally, but to be nonmutagenic in Salmonella. We have tested these two compounds for their ability to induce sister chromatid exchanges in the colonic epithelium of mice, the same tissue in which they induce nuclear anomalies when administered by the same route. One, 4-cholesten-3-one, induced sister chromatid exchanges whereas the other, 5-alpha-cholestan-3-one did not, even at the maximum feasible dose. The results suggest that 4-cholesten-3-one is more likely to be a significant factor in human colon cancer than the 5-alpha analog.

Screening for colon carcinogens--a new strategy.

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.

Induction of sister chromatid exchanges in murine colonic tissue.

As cancers of the large bowel arise primarily in the epithelial cells of the colon, measurement of genetic damage induced in those cells may aid in elucidating contributing factors in the etiology of the disease. Accordingly, procedures for the in vivo measurement of sister chromatid exchanges (SCE) have been adapted for use with colonic cells. Agar-coated 5-bromo-2'-deoxyuridine (BrdUrd) tablets were implanted (s.c.) in mice and colonic tissue prepared for measurement of SCE 48 hr later. When mice were treated by intraperitoneal injection with 20 mg/kg 1,2-dimethylhydrazine (DMH), a colon carcinogen, a significant increase over control SCE frequencies was found when treatment with carcinogen occurred in the interval from 14 hr before to 36 hr following BrdUrd administration. Treatment with DMH 2 hr following implantation of BrdUrd produced a dose-dependent increase in SCE frequencies, with the highest dose (20 mg/kg) resulting in frequencies approximately twice those of controls. Dimethylhydrazine was not found to be effective in inducing SCE in bone marrow cells of mice. As SCE can be induced by carcinogens and mutagens, measurement of SCE in colonic epithelium may be useful in identifying potential colon carcinogens.

The use of primary rat hepatocytes to achieve metabolic activation of promutagens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase mutational assay.

A method is described in which primary rat hepatocytes have been cocultured with Chinese hamster ovary (CHO) cells to provide metabolic activation of promutagens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fischer-344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum-free medium. The survival and 6-thioguanine-resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B1 (AFB1) 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(A)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB1 was approximately 8 days. Primary cell-mediated mutagenesis may be useful in elucidating metabolic pathways important in the production and detoxification of genotoxic products in vivo.

A critical review of the literature on nitrobenzene toxicity.

This literature review encompassing information available through 1980 and limited coverage in 1981, emphasizes results useful in assessing the potential toxic effects of nitrobenzene to man. Nitrobenzene exposure in man or experimental animals is most often associated with methemoglobinemia. Histopathologic changes also are observed in the hemato-lymphoreticular system, central nervous system, and liver. In addition, lesions have been reported in adrenals and testes. No information was found on carcinogenic or teratogenic potential, fertility, or reproductive effects of nitrobenzene. Results from Ames Salmonella assay are negative but test procedures are questionable; metabolites give positive results. Metabolism of nitrobenzene involves either oxidation or reduction yielding p-aminophenol and p-nitrophenol, and other reduced intermediates. From the foregoing, several aspects of nitrobenzene toxicity have been identified which warrant further study. Recommendations are made for chronic and subchronic exposure of test animals via inhalation to assess various toxicological endpoints. In addition, the relationship of nitrobenzene metabolism to its toxicity needs to be established. Genotoxic effects of nitrobenzene also need study.

Cytotoxicity and mutagenicity of dinitrotoluenes in Chinese hamster ovary cells.

Technical grade dinitrotoluene (DNT), a mixture composed predominantly of 2,4- and 2,6-DNT but containing lesser amounts of 2,3-, 2,5-, 3,4- and 3,5-DNT, has been shown to be a hepatocarcinogen in rats, The mutagenicity of these compounds has been evaluated using the CHO/HGPRT system, a quantitative mammalian somatic cell mutational assay. Dinitrotoluenes were tested for their ability to induce mutation to 6-thioguanine (TG) resistance in the presence and absence of microsomal preparations (PMS) from rats pretreated with the mixed function oxidase inducer Aroclor 1254. A marked difference in cytotoxicity of the isomers was observed. However, neither technical grade DNT nor any of the purified isomers resulted in a significant increase in the TG-resistant fraction of surviving cells, with or without added PMS.

The mutagenicity of dinitrotoluenes in Salmonella typhimurium.

The mutagenicity of technical grade 2,4-dinitrotoluene (DNT), a mixture composed predominantly of 2,4-DNT (76.5%) and 2,6-DNT (18.8%) but containing 3,4-, 2,3-, 2,5- and 3,5-DNT, and the 6 isomers of DNT has been determined in 3 assays with Salmonella typhimurium. The mixture and the individual isomers of DNT were found to be mutagenic in the Ames' Salmonella/microsome test, particularly in strains responding to frame-shift mutagens; 3,5-DNT was found to be the most effective isomer in inducing reversion to histidine prototrophy in S. typhimurium strains TA98 and TA1538. Similar results were obtained with a quantitative reversion assay using strain TA98; 3,5-DNT was the most mutagenic of the compounds examined, particularly in the absence of post-mitochondrial supernants of rat-liver homogenates. While all isomers and the mixture of DNTs increased the 8-azaguanine-resistant fraction of S. typhimurium TM677 to some degree, the greater mutagenicity of 3,5-DNT compared to other isomers was not evident in the forward mutational assay.

Access systems of heavy construction vehicles: Parameters, problems and pointers.

As part of an investigation of accidents occuring during mounting on and dismounting from heavy construction equipment a study was conducted of operator behaviour during mounting and dismounting. Measurements of access systems of representative vehicles were compared with those from various Standards. In most instances the vehicles did not conform. Recommendations are made for improvements in the safety characteristics of the access. These recommendations are in three classes, namely; changes and developments in procedures and practices, retrofit of existing equipment, and changes in design philosophy. The need for maintaining three point contact at all times during mounting and dismounting is emphasised.

Stability of activating systems for in vitro mutagenesis assays: enzyme activity and activating ability following long-term storage at - 85 degrees C.

Activating systems for in vitro mutagenesis assays are commonly prepared and stored at low temperature until required. The objective of the studies reported here was to determine the long-term stability of activating systems stored at - 85 degrees C. A broad range of microsomal enzymes in the postmitochondrial supernatant (PMS) and the microsomal fraction of livers from Aroclor 1254 treated rats were studied in conjunction with the ability of these fractions to catalyse the conversion of dimethylnitrosamine (DMN) and benzo(a)pyrene (B(a)P) to products mutagenic to Chinese hamster ovary (CHO) cells and Salmonella typhimurium TM677. Biphenyl-2- and biphenyl-4-hydroxylase showed a rapid decline in activity on storage, epoxide hydratase activity increased with storage and other enzyme activities studied were relatively stable for up to 32 weeks. No consistent trends in the ability of either the microsomes or the PMS to catalyze DMN or B(a)P induced mutation were observed for up to 12 weeks with CHO cells and 24 weeks with bacteria. It is concluded that low temperature storage of activating systems is an acceptable procedure. However, the results also indicate that certain enzyme activities change during storage, suggesting that aberrant results may be obtained when stored activating systems are used in in vitro tests to screen for mutagens.

Mutagenicity of dimethylnitrosamine and ethyl methanesulfonate as determined by the host-mediated CHO/HGPRT assay.

Host-mediated assays have been developed to allow determination of the mutagenic potential of promutagens and procarcinogens which require metabolic activation to exert their effects on indicator organisms. We report here the development of the host-(mouse)-mediated CHO/HGPRT system using the procarcinogen dimethylnitrosamine (DMN) as a model agent. Using a 2--h treatment time, we observed a linear dose-response relationship up to 250 mg of DMN per kg body weight. At 100 and 500 mg/kg DMN, mutation induction increased with time up to at least 6 h. DMN was not mutagenic when tested in vitro. Athymic (nude) mice, their phenotypically normal littermates, or BALB/c mice of both sexes were found to be suitable as hosts. A time- and dose-dependency of induced mutation frequency by a direct-acting agent, ethyl methanesulfonate (EMS), was observed in both the in vitro and the host-mediated assays.