Comparison of the in vitro mutagenicity and metabolism of dimethylnitrosamine and benzo[a]pyrene in tissues from inbred mice treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls.

Homogenates of liver, lung, kidney, stomach, small intestine and colon from 8 strains of mice were compared for their ability to metabolize benzo[a]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains CF1, AKR/J, AU/SsJ, DBA/2J, SWR/J, A/J, C3H/HeJ, and C57BL/6J were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. The effects of these drugs on organ weight and on the amounts of DNA, S-10 protein, and microsomal protein per unit weight of tissue are reported. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. For each organ there was an optimal balance between amount of tissue homogenate and concentration of test compound for maximal yield of revertants. A sensitive radiometric assay of DMN demethylase (DMND) is described which permits measurement of the enzyme in liver, lung and kidney. DMN at 1 mM is used as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured in all tissue using BP as substrate. AR and MC are very good inducers of AHH activity in livers of mice classified as aromatic hydrocarbon responsive, but not in those classified as hydrocarbon nonresponsive. Responsiveness is strain-specific and genetically regulated. Metabolism of BP to mutagens by liver homogenates was correlated with extent of AHH induction. This dimorphism of response of AHH to inducers was present, but less pronounced, in non-hepatic tissues. Basal activities of AHH and DMND were correlated in livers and lungs from untreated mice. DMND activities were increased less than 2-fold by PB, MC or AR treatments. Metabolism of DMN to mutagens was not closely correlated with DMND activities. Strain of mouse, type of tissue and test substance are important variables in assessing the potential effect of microsomal enzyme-inducing agents on the metabolism of mutagenic substances.     

Mutagenicity and metabolism of dimethylnitrosamine and benzo[alpha]pyrene in tissue homogenates from inbred Syrian hamsters treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls.

There are significant differences between mice and hamsters in polycyclic hydrocarbon and nitrosamine metabolism. Homogenates of liver, lung and intestinal mucosa from 6 strains of Syrian golden hamster were compared for their ability to metabolize benzo[alpha]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains MHA/SSLak, LSH/SlLak, CB/SsLak, PD4/Lak LHC/Lak and Lak:LVG (SYR) were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagans. Dimethylnitrosamine demethylase (DMND) was assayed using 1 mM DMN as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured using benzo[alpha]pyrene as substrate. MC does not induced AHH activity in hamster liver, but is an excellent inducer of enzymes converting BP to mutagens. This lack of correlation between increased AHH activity and increased metabolism of BP to mutagen in liver is in marked contrast to correlations seen in mice. MC induces AHH in hamster lung and intestinal mucosa. AR induces AHH in liver, lung and intestinal mucosa. Activity of DMND in liver is not affected by treatment of hamsters with BP or AR, but is repressed approx. 30% by treatment with MC. Activity of DMND and conversion of DMN to mutagen are correlated (r = 0.59) in hamster liver. Microsomes of hamster liver are more effective than those from mouse in converting DMN to mutagen, despite similar DMND activities in livers from the two species.     

Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100

Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella typhimurium strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver S9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of S9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60% of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without S9. BP, 3MC and 3MCE were not mutagenic in either strain without S9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the S9 preparation revealed decreased AHH activity with increase in Se concentration. The decreased mutagenicity and AHH activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as Na2SO3). Inhibition of AHH activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. typhimurium TA98 and TA100.     

The aromatic hydrocarbon receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and variable levels of induced aryl hydrocarbon hydroxylase activity in clones of mouse hepatoma cells

Induction of aryl hydrocarbon hydroxylase (AHH) activity was studied in clones and subclones of mouse hepatoma (Hepa-lcl) cells. When maximally induced, one clone had significantly lower (p less than 0.005), two had approximately the same, and two had significantly higher (p less than 0.005) levels of AHH activity compared with Hepa-lcl. The maximal level of induced activity, relative to the parent population, in two clones chosen for further analysis was 0.14 +/- 0.09 for clone 1 (Hs-1) and 0.94 +/- 0.28 for clone 9 (Hs-9). These relative levels were stable over a period of 10 months and were similar when activity was induced either with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz[a]anthracene. Subclones of Hepa-lcl cells, derived from the Hs-9 clone, also demonstrated variation in induced AHH activity. When maximally induced with TCDD, six subclones had significantly lower AHH activity (p less than 0.005), two had approximately the same, and one had significantly higher levels (p less than 0.005) compared with the progenitor Hs-9 population. Comparative analysis of Ah receptor characteristics in two unselected clones of Hepa-lcl with significantly different levels of AHH activity demonstrated that there was no apparent correlation between relative level of induced AHH activity and (i) total quantity of Ah receptor (cytosol and nuclear), (ii) receptor affinity for TCDD and number of receptor sites in each cell, (iii) subcellular distribution of [3H]TCDD, or (iv) specificity and saturable nature of binding. Coordinate measurement of the concentration of nuclear receptor and absolute induced AHH activity in Hepa-lcl and its clones had a positive correlation (r = 0.79).     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Genetics of aryl hydrocarbon hydroxylase induction in mice: Response of the lung to cigarette smoke and 3-methylcholanthrene

When mice from different inbred strains are injected intraperitoneally with 3-methylcholanthrene (MC), the activity of aryl hydrocarbon hydroxylase (AHH) rapidly increases in livers of some strains but not others. AHH plays a role in the metabolism of polycyclic hydrocarbons. Alleles at a small number of loci account for most of the variation in inducibility of hepatic AHH among mice, when MC is used as the inducing agent. Cigarette smoke is a common source of carcinogenic polycyclic hydrocarbons in the environment. Since some of the hydrocarbons in cigarette smoke are metabolized by AHH, the activity of AHH in tissues may affect the carcinogenicity of smoke in those tissues. The purpose of these experiments was to see whether induction of AHH in lung in response to cigarette smoke is regulated by the same genes that regulate induction of AHH in liver in response to MC. Mouse strains AKR/J and C57L/J and six recombinant inbred (RI) lines derived from them were tested for the response of AHH in lung and liver to parenteral MC or inhalation of cigarette smoke. Inducibility (the ratio of MC-induced AHH activities to basal AHH activities) in liver from MC-treated RI lines is bimodal and compatible with Mendelian segregation of genes at a small number of loci. Increased activities of AHH in MC-treated liver are associated with increased ability to metabolize BP and whole smoke condensates to mutagens detected by Salmonella typhimurium TA1538. Inducibility of AHH in lung in response to MC is not bimodal, and no definite conclusion about the number of loci can be made. When actual levels of AHH activity are considered, following the administration of MC as inducing agent, there is a correlation (r=0.89, p<0.01) between AHH levels in liver and lung, suggesting that some genes affecting liver also affect lung. Basal and MC-induced AHH levels in lung are also correlated (r=0.86, p<0.01). Mice with high basal activities have two to threefold higher levels of AHH after MC treatment than do mice with low basal activities. Induction of AHH in pulmonary tissues occurs in all mice after either parenteral MC or smoke inhalation. In contrast to MC treatment, AHH activities in lungs following smoke inhalation are not correlated with AHH levels in liver after MC (r=0.49) and are only weakly correlated with basal (r=0.66, 0.05相似文献   

Spontaneous Conversion of Nontransformed Avian Sarcoma Virus-Infected Rat Cells to the Transformed Phenotype

Normal rat kidney (NRK) fibroblasts were infected with the Schmidt-Ruppin strain (SR-D) of avian sarcoma virus (ASV) and cloned 20 h after infection without selection for the transformed phenotype. Most infected clones initially exhibited the flat, nontransformed morphology that is characteristic of uninfected NRK cells. In long-term culture, however, the majority of the SR-D NRK clones began segregating typical ASV-transformed cells. Transforming ASV could be rescued by fusion with chicken embryo fibroblasts from most of the infected clones tested. Three predominantly flat, independently infected clones were further analyzed by subcloning 8 to 10 weeks after infection. Most flat progeny subclones derived at random from two of these "parental" SR-D NRK clonal lines did not yield virus upon fusion with chicken embryo fibroblasts, although a nondefective transforming ASV was repeatedly recovered from the parental clones. This observation suggested that most, but not all, daughter cells in these SR-D NRK clones lost the ASV provirus after cloning. The progeny of the third independent parental cell clone, c17, gave rise to both flat and transformed subclones that carried ASV. In this case, ASV recovery by fusion and transfection from the progeny subclones was equally efficient regardless of the transformation phenotype of the cells. The 60,000-dalton phosphoprotein product of the ASV src gene was, however, expressed at high level only in the transformed variants. The results of a Luria-Delbruck fluctuation analysis and of Newcombe's respreading test indicated that the event leading to the spontaneous conversion to the transformed state occurred at random in dividing cultures of these flat ASV NRK cells at a rate predicted for somatic mutation.     

6-Amino-chrysene, a potent inhibitor of transferase activity in single living RTG2 cells

Previous reports on the inhibitory effect of 6-amino-chrysene (6AC) on benzo(a)pyrene (BP) metabolism using single living cells have suggested that aryl hydrocarbon hydroxylase (AHH) is not the only pathway for 6AC metabolism. We present here results demonstrating that direct glucuronidation may constitute an alternative pathway for 6AC elimination. First, we describe the conjugate of 6AC to UDP-glucuronic acid (UDPGA) in solution. We performed competition experiments between 6AC and monohydroxy BP, which are known to be good substrates for glucuronic transferase (GT), in RTG2 cells, using microspectrofluorimetry. Because of intracellular accumulation of fluorescent metabolites during BP metabolism, RTG2 cells can be used as a tool for simultaneous study of AHH and GT activities. When RTG2 cells have been simultaneously treated with BP and 6AC, GT appeared to be a more specific target for 6AC than AHH in these cells. Therefore, 6AC can be expected to act as a more specific inhibitor for GT than for AHH activity.     

Carcinogen mmetabolism and DNA adducts in human lung tissues as affected by tobacco smoking or metabolic phenotype: a case-control study on lung cancer patients

Individual variations in activity of pulmonary enzymes that metabolize tobacco-derived carcinogens may affect an individual's cancer risk from cigarette smoking. To investigate whether some of these enzymes (e.g., cytochrome P450IA-related) can serve as markers for carcinogen-induced DNA damage accumulating in the lungs of smokers, non-tumorous lung tissue specimens were taken during surgery from middle-aged men with either lung cancer (n = 54) or non-neoplastic lung disease (n = 20). Phase I (AHH, ECDE) and phase II (EH, UDPGT, GST) enzyme activities, glutathione and malondialdehyde contents were determined in lung parenchyma and/or bronchial tissues; some samples were analyzed for DNA adducts, using ³²P-postlabeling.

Data analysis of subsets or the whole group of patients yielded the following results. (1) Phase I and II drug-metabolizing enzyme (AHH, EH, UDPGT, GST) activities in histologically normal surgical specimens of lung parenchyma were correlated with the respective enzyme activities in bronchial tissues of the same subject. (2) In lung parenchyma, enzyme (AHH, ECDE, EH, UDPGT) activities were significantly and positively related to each other, implying a similar regulatory control of their expression. (3) Mean activities of pulmonary enzymes (AHH, ECDE) were significantly (2- and 7-fold, respectively) higher in lung cancer patients who had smoked within 30 days before surgery (except GST, which was depressed) than in cancer-free subjects with a similar smoking history. (4) In the cancer patients, the time required for AHH, EH and UDPGT activities to return to the level found in non-smoking subjects was several weeks. (5) Bronchial tree and peripheral lung parenchyma preparations exhibited a poor efficiency in activating promutagens to bacterial mutagens in Salmonella. However, they decreased the mutagenicity of several direct-acting mutagens, an effect which was more pronounced in tissue from recent smokers. GSH concentration and GST activity were positively correlated with mutagen inactivation in the same sample. (6) In recent smokers, AHH activity in lung parenchyma was positively correlated with the level of tobacco smoke-derived DNA adducts. (7) Pulmonary AHH and EH activity had prognostic value in tobacco-related lung cancer patients. (8) An enhanced level of pro-oxidant state in the lungs was associated with recent cigarette smoking. Malondialdehyde level in lung parenchyma was associated with the degree of small airway obstruction, suggesting a common free radical-mediated pathway for both lung cancer induction and small airway obstruction.

These results demonstrate the pronounced effect of recent cigarette smoke exposure on pulmonary xenobiotic metabolism and lipid peroxidation and lend further support to the hypothesis that the inducibility of pulmonary AHH activity (cytochrome P450IA1 levels) in tobacco smokers is associated with lung cancer risk. Results on DNA adducts in smokers' lung tissue may help to explain why a certain metabolic phenotype accumulates more DNA damage in lung cells.     

Metabolism of benzo[A]pyrene and the related enzyme activities in hamster embryo cells.

The rate of metabolism of benzo[a]pyrene (BP) and changes in related enzyme activities in cultured hamster embryo cells during successive subculture were studied. The activity of aryl hydrocarbon hydroxylase (AHH) was the highest when embryo cells were first dispersed in tissue culture flasks and decreased during subsequent passages. On the other hand, UDP-glucuronyl transferase activity increased gradually during successive subculture. Treatment of the cells with 13 nmol/ml of benz[a]anthracene (BA) for 24 h increased the activity of AHH but not that of UDP-glucuronyl transferase. The metabolism of BP was measured in cells of the passages 1, 3 and 7; metabolism of BP was most efficient in cells in passage 3 and their formation of glucuronic acid conjugates of BP, one of the major metabolites found in the medium, was 3- and 10-fold more than those of cells in passages 1 and 7, respectively. Analysis of BP-metabolites extracted from the medium with ethylacetate showed that the main metabolites were 9,10-diol and 7,8-diol. Phenols and quinones were released by treatment of the medium with beta- glucuronidase and their amounts were larger than those of diols at all passages. These results show that in hamster embryo cells in early passage, BP is metabolized to conjugates of phenols with glucuronic acid.     

Derivation of Tk- Clones from Revertant Tk+ Mammalian Cells

In order to obtain a large collection of Chinese hamster cell clones defective in thymidine kinase (TK(-)), BrdU(r) selection experiments have been performed on wild-type and revertant TK(+) cell lines. No clones (< 10(-9)) were obtained from the wild-type TK(+) cell line by single-step selection. In contrast, revertant TK(+) clones readily gave rise to stable TK(-) derivatives (1 - 2 x 10(-4)). Both wild-type and revertant TK(+) clones spontaneously yielded 8-AG(r) colonies with the same frequency (1 - 5 x 10(-6)), suggesting that the differences between wild-type and revertant cell lines specifically affected selection of the TK(-) phenotype. The increased frequency of TK(-) clones reflects perhaps the number (ploidy) or character of the autosomal TK loci in TK(+) revertants, or perhaps the mechanisms which regulate expression of the TK genes. Several mutagens, EMS, MNNG and UV, stimulated the TK(+) revertants' frequency of TK(-) subclones only slightly (< 3-fold). Biochemical and genetic data indicated that the TK(-) clones derived from one revertant are phenotypically different. The phenotypes displayed by these cell lines are stable and do not depend upon the continued presence of the selective agent.     

The induction of thymidine- and IUdR-resistant variants in P388 mouse lymphoma cells by x-rays, UV and mono- and bi-functional alkylating agents

Dose-response curves for “mutation” to resistance to 5-iodo-2-deoxyuridine (IUdR) and excess thymidine (TdR) in P388 mouse lymphoma cells have been established after exposure of these cells to six chemical mutagens, UV |and| ionising radiations. The dose-response curves for all mutagens in both selective system show considerable similarities when induced mutation frequencies are plotted against survival. Expression time for both types of variants, IUdR^r and TdR^r, are similar, i.e. maximum frequencies are reached by 48 h and there is no fall in variant frequency at late expression times up to 144 h. Over the range of survival levels studied there appears to be little or no dependence of expression time on dose of mutagen. Some loss of mutants after high doses (i.e. at low survival levels) was observed due to the fact that a significant proportion of both TdR^r and IUdR^r clones were more sensitive to the mutagens than the wild-type population. The similarities in induced dose-response curves for different mutagens suggest that the mutants have a common origin, probably an error in repair, but it seems unlikely that errors in “cut and patch” repair are responsible. A comparison of spontaneous frequencies of IUdR^r and TdR^r variants suggests that IUdR is mutagenic in P388 cells.     

Genetic transformation of somatic cells. VII. The loss of the transformant phenotype is accompanied by both stable and unstable changes in DNA

From 6 clones of Chinese hamster cells varying in the rate of the loss of transformant phenotype and containing a thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1), 25 subclones negative in thymidine kinase (TK-) were isolated on a medium with 50 micrograms/ml 5-bromodeoxyuridine (BrdU). A study was made of the frequency of spontaneous reversions to the TK+ phenotype in cell populations of BrdU-resistant subclones, and of the transforming activity (upon transformation of TK- cells of A238 clone to the TK+ phenotype) of DNA preparations from a row BrdU-resistant subclones. In 7 of 11 BrdU-resistant subclones the TK- phenotype is associated with changes reducing significantly the transforming activity of DNA. Some of these alterations are stable and undergo no spontaneous reversion, while the other ones are unstable, being reversed or suppressed at a high frequency. BrdU-resistant subclones produced from clones more stable in transformant phenotype are on the whole more stable in the TK- phenotype than BrdU-resistant subclones from the clones with the high rate of the loss of the TK+ phenotype.     

Biochemical basis for the acquisition of resistance to benzo[a]pyrene in clones of mouse liver cells in culture

In a Namru mouse liver epithelial cell strain designated NMuLi, aryl hydrocarbon hydroxylase (AHH) activity peaked at 12 h post-induction with 1 μg/ml of benzo(a)pyrene (BaP) in both confluent and growing cells. Maximal levels of AHH activity were reached on day two post-plating. This induced activity was inhibited in vitro 78% by gassing the incubation mixture with carbon monoxide for 15 s, and inhibited 93% by addition of 40 μg/ml of 7,8 benzoflavone(BF).Induced AHH levels were higher in epithelial clones that were sensitive to the toxicity of BaP than in resistant clones. The survival fraction of clones from NMuLi and of subclones derived from a sensitive clone of NMuLi after BaP treatment was a negative exponential function of the maximal induced AHH activity in the clones.One of the clones, NMuLi cl 8, was extremely susceptible to the toxic effects of BaP, the ±(trans)-7α, 8β-dihydroxy-7,8-dihydro-BaP(7,8-diol), and the (±)-7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro-BaP (diolepoxide), known metabolites of BaP. The toxicity of BaP and the 7,8 diol to this clone was inhibited by BF, suggesting that these cells possessed an enzyme activity inhibitable by BF that could epoxidize BaP to the 7,8 oxide and then epoxidize the resultant 7,8 diol to the diol-epoxide. Another clone derived from NMuLi, clone 7, was relatively resistant to the toxic effects of BaP and the 7,8-diol, but still extremely susceptible to the toxic effects of the diol-epoxide. The slight toxicity to BaP in this clone was inhibited by BF, but the toxicity of the 7,8-diol to this clone was not inhibited by BF. A typical cytochrome P450 inhibitor, metyrapone, had no effect on the toxicity of BaP, the 7,8-diol, or the diol-epoxide to either clone 7 or clone 8.The results suggest that these liver cells possess two enzymes that play some role in polycyclic hydrocarbon-induced toxicity. Enzyme A, a BaP-inducible enzyme that is inhibitable by BF, efficiently metabolizes BaP to the 7,8-diol and the 7,8-diol to the diol-epoxide. It is responsible for most of the hydrocarbon toxicity. Enzyme B is not inhibitable by BF and metabolizes the 7,8-diol less efficiently to the diol-epoxide or efficiently to other, less toxic products.     

Transformation-defective mutants of feline sarcoma virus which express a product of the viral src gene.

Mink cell cultures infected with the Snyder-Theilen strain of feline sarcoma-leukemia virus were cloned from single cells under conditions favoring single virus-single cell interactions. The primary colonies included (i) typical feline sarcoma virus (FeSV)-transformed nonproducer clones, one of which segregated revertants, and (ii) FeSV-infected, phenotypically normal clones, three of which spontaneously converted to the transformed phenotype. The revertants and spontaneous transformants were compared with parental and sister clones expressing the opposite phenotype. Transformed subclones formed colonies in agar, were tumorigenic in nude mice, and failed to bind epidermal growth factor, whereas flat sister subclones were indistinguishable from uninfected mink cells in each of these assays. Sister subclones derived from the same infectious event contained FeSV proviruses integrated at the same molecular site, regardless of which phenotype was expressed. One revertant clone, however, lacked most FeSV proviral DNA sequences but retained terminal portions of the FeSV genome which persisted at the original site of proviral DNA insertion. Two flat subclones expressed viral RNA and the phosphorylated "gag-x" polyprotein (pp78gag-x) encoded by the gag and src sequences of the FeSV genome. Both of these clones were susceptible to retransformation by FeSV. Although unable to induce foci, the viruses rescued from these cells contained as much FeSV RNA as the focus-forming viruses rescued from transformed sister subclones and could be retransmitted to mink cells, again inducing FeSV gene products without signs of morphological transformation. We conclude that these FeSV genomes represent transformation-defective mutants.     

Fitness effects of two facultative endosymbiotic bacteria on the pea aphid,Acyrthosiphon pisum,and the blue alfalfa aphid,A. kondoi

The effects of two bacterial endosymbionts, designated PASS and PAR, were evaluated on the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera:Aphididae), in which they occur facultatively, and on the blue alfalfa aphid, A. kondoi Shinji, in which these bacteria have not been found in natural populations. Subclones of pea aphids and blue alfalfa aphids, derived from parent aphid clones that did not contain PASS or PAR, were infected with one or both bacteria, generating PASS- and/or PAR-positive subclones with minimal genetic differences from the parent clones. Under laboratory conditions at 20 °C, PAR consistently reduced the fecundity (by between 19 and 60%) of subclones derived from three different parent pea aphid clones on bur clover, Medicago hispida Gaertn. PAR had intermediate effects on pea aphids reared on sweet pea, Lathyrus odoratus L., and had no significant effect on pea aphids on alfalfa, Medicago sativa L. The effect of PASS was either neutral or negative, depending on parent clone as well as host plant. Also at 20 °C, PASS reduced fecundity (70–77%) and longevity (40–48%), and increased the age of first reproduction (by up to 1.5 days) of blue alfalfa aphid reared on alfalfa and clover. PAR had a less dramatic effect (e.g., 30–39% reduction in fecundity) on these traits of blue alfalfa aphid. In contrast, PAR and PASS increased the fitness of pea aphid subclones of one parent clone reared for three generations at 25 °C on each of the three test plants. Without facultative bacteria, fecundity of the parent clone was reduced to a mean total of < 6 offspring per adult at this elevated temperature, but with PASS or PAR, mean total fecundity of its subclones was > 35. However, this ameliorative effect of facultative bacteria at 25 °C was not found for two other sets of parent clones and their derived subclones. Alate production in pea aphids was significantly increased in large populations of two PASS- and PAR-positive subclones relative to their parent clones. Attempts to transmit PASS or PAR horizontally, i.e., from aphid to aphid via feeding on host plants (bur clover), were unsuccessful.     

Metabolic activation of promutagens, detectable in Ames' Salmonella assay, by 5000 X g supernatant of rat ventral prostate

Induction of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin-O-deethylase (7-EOD) activities as well as of benzo[a]pyrene (BP) metabolite formation in rat prostatic microsomes has been demonstrated after treatment with beta-naphthoflavone (BNF). The capacity to convert promutagenic compounds to ultimate mutagenic metabolites in the Ames' Salmonella assay by 5000 X g supernatant of rat ventral prostate was investigated. Male rats were treated with BNF, polychlorinated biphenyls (PCB; Arochlor 1254), phenobarbital (PB) and the vehicle, corn oil. PCB or BNF pretreatment increased the AHH- and 7-EOD activities 100-200-fold in the rat prostate 5000 X g supernatant (S-5 fraction). Epoxide hydrolase (EH) and glutathione-S-transferase (GST) activities were not affected while UDP-glucuronosyltransferase (UDP-GT) was increased 2.2- and 2.5-fold by PCB and BNF, respectively. PB did not significantly affect any of the enzyme activities measured. A dose-dependent increase in mutagenic response versus amount of 5000 X g supernatant and promutagen (aflatoxin B1 (AFB), 2-aminofluorene (2-AF), BP) was observed. The most pronounced activation was obtained with S-5 fraction from BNF- or PCB-treated rats. The great sensitivity of prostatic AHH to certain inducers and the capacity of the prostate to produce mutagenic metabolites might be of importance for initiation of prostatic cancer by environmental factors.     

Association of steroid sulfatase with one of the arylsulfatase C isozymes in human fibroblasts

When arylsulfatase C, a microsomal membrane-bound enzyme, is assayed with its natural substrates, the 3-beta-hydroxysteroid sulfates, it is also known as steroid sulfatase. Whether arylsulfatase C and steroid sulfatase are identical enzymes or not, however, has long been disputed. We now report that two electrophoretic variants of arylsulfatase C occur in normal human fibroblasts: one has a single anodic band of activity, "s," and the other has an additional faster migrating band, "f". The two types, s and "f + s", occur in cells from either sex. When fibroblast strains with the f + s forms of arylsulfatase C were cloned, two types of primary clones were always obtained: s and f + s. A single f band was never seen. When these primary clones were subcloned, however, the arylsulfatase C phenotype remained unchanged: primary s clones gave rise to s subclones and f + s clones to f + s subclones only. Therefore, these forms were clonal in origin and demonstrated a novel inheritance pattern in human cultured cells. The appearance of increasing amounts of the f band was correlated with up to 4-fold increase of arylsulfatase C activity, whereas the steroid sulfatase activity remained constant, thus demonstrating that arylsulfatase C was not identical with steroid sulfatase activity. Polyclonal antibodies raised against the s form immunoprecipitated activities of the s form of arylsulfatase C and steroid sulfatase but not the f form of arylsulfatase C. Therefore, we conclude that only the s form of arylsulfatase C is immunologically related to steroid sulfatase so that arylsulfatase C per se is not necessarily identical with steroid sulfatase. In addition, a novel form of genetic heterogeneity of isozymes in human fibroblasts is demonstrated.     

Vitamin K1 amplification of benzo(a)pyrene metabolism in chick embryos

Seventeen-day-old chick embryos were used as a test system to assess the effect of vitamin K1(K1) on benzo(a)pyrene (BP) metabolism as measured by the induction of arylhydrocarbon hydroxylase (AHH) and cytochrome P-450 and the levels of glutathione (GSH) and glutathione S-transferase (GST) in liver. Twenty-four hours after injection of BP into the air sac there was a sharp rise in AHH and P-450 and a drop in GSH. When K1 was injected 24 hr prior to BP there was a decrease in GST activity as compared with the control plus an augmented increase in AHH induction. This augmentation in BP metabolism (Phase I) together with a concomitant decrease in at least one mechanism of Phase II conjugation is in keeping with other evidence that K1 can play an adjuvant role in BP induced mutagenicity and carcinogenicity. Ubiquinone has a much lesser effect on BP metabolism than does K1 in equimolar concentration.     

Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon

The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon hydroxylase (AHH) activity and binding levels of BP to macromolecules were higher in the descending colon when compared to other segments. The major metabolites of BP, extractable with ethylacetate, were quinones, tetrols, 7,8-diol and a peak containing 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene and 7,8,9-trihydroxy-7,8-dihydrobenzo(a)pyrene. The binding levels of BP to DNA and protein in the explant was lowered by co-incubation with 7,8-benzoflavone (7,8-BF) (3.6 and 18.0 μM), a known inhibitor of AHH, and with disulfiram (100 μM), an anti-oxidant. The absence of vitamin A in the media also resulted in a lower level of BP binding to DNA and protein and in lower activity of AHH. Pretreatment with known inducers of AHH such as phenobarbital (PB) or benz(a)anthracene (BA), did not have any significant effect on the binding levels of BP to DNA or on the AHH activity. of the bile acids investigated only taurodeoxycholic acid significantly increased the binding level of BP to DNA.