Campus drinking: an epidemiological model.

The drinking behaviours of college students have posed significant public health concerns for several generations. However, the dynamics of campus drinking have not been analysed using mathematical models. An epidemiological model capturing the dynamics of campus drinking is used to study how the 'disease' of drinking is spread on campus. The model suggests that the reproductive numbers are not sufficient to predict whether drinking behaviour will persist on campus and that the pattern of recruiting new members plays a significant role in the reduction of campus alcohol problems. In particular, campus alcohol abuse may be reduced by minimizing the ability of problem drinkers to directly recruit non-drinkers.

The effects of different levels of dietary restriction on aging and survival in the Sprague-Dawley rat: implications for chronic studies.

A study was undertaken to determine the effects of incremental levels of dietary restriction (DR) in rats. Survival, growth, reproductive, and dietary intake (DI) variables were monitored in a chronic study in which male Sprague Dawley (SD) rats (NCTR colony) were fed their ration ad libitum (AL), or DR. The main objectives were to determine if low levels of DR could be used to increase the survival rate of SD rats in the chronic bioassay, and to identify the survival characteristics of a long-lived SD rat strain (NCTR colony). The average life span of AL rats was 115 months. At 104 weeks on study (110 weeks of age), the survival rate for the AL and 10%, 25%, and 40% DR groups was 63.4, 87.5, 87.5, and 97.5%, respectively. The largest increase in survival (24.1%) occurred between AL and 10% DR, indicating that very low levels of DR have a significant effect on survival. Whole-body, liver, prostate, and epididymis weights and body length were decreased by DR, whereas brain weight, testicular weight, and skull length were not altered by DR. Rats from the NCTR colony were found to be ideal for chronic studies because they are much longer-lived than other SD stocks. Although the 104-week survival rate for these SD, non-obese AL rats exceeds the FDA's "Redbook" survival guideline (> 50%) for chronic bioassays, the use of DR is advocated because it reduces individual variability in body weight.

Activity profile of glutathione-dependent enzymes and respiratory chain complexes in rats supplemented with antioxidants and treated with carcinogens.

Appropriate dietary interventions may reduce the potentially damaging effects of free radicals generated during metabolism and various physiological conditions. We have investigated the effects of dietary vitamins C, E, beta-carotene, or selenium (Se) on the activity of endogenous antioxidant enzymes and respiratory chain complexes in rats exposed to 7,12-dimethylbenz[a]anthracene (DMBA), a mammary carcinogen and bleomycin (BLM), an antineoplastic drug. These agents are known to generate DNA-reactive species during their metabolism, which may enhance oxidative stress in cells. Female Fischer 344 rats aged 4 months were given antioxidant supplements singly or as a mixture 2 weeks prior to mutagen treatments; antioxidant supplementation continued for an additional 4 weeks. In rats treated with mutagens, the antioxidant intake lowered the activity of Se-dependent glutathione peroxidase (Se-GPx) in liver cytosolic and mitochondrial fractions, compared to activity in rats treated with mutagens alone. However, the vitamins, but not Se supplement, persistently increased Se-GPx activity in untreated control animals. Treatment of animals with mutagen raised K(m) value of Se-GPx and this correlated with an increase in V(max). However, Se intake, either singly or mixture, significantly reduced K(m) value in mutagen-treated and untreated rats in both fractions. Se intake increased glutathione S-transferases (GST) activity (P < 0.05) in both liver fractions of mutagen-treated and untreated animals. Similar response was seen in Se-independent GPx. Since GST-alpha possesses Se-independent GPx activity, the enhanced effect observed in GST activity may be due, in part, to increased activity in Se-independent GPx. Also, selenium or the antioxidant vitamin supplementation increased the activity of all four respiratory chain complexes in untreated rats. Although BLM treatment significantly increased the activity of electron transport complexes III and IV, selenium or the vitamin supplements modulated the responses. These results indicate that the intake of dietary vitamins or Se enhances antioxidant capacity in chemically exposed animals compared to animals receiving antioxidants alone. Furthermore, in addition to being an enhancer of the catalytic function of glutathione peroxidase, selenium may directly play a role as an antioxidant.

Influence of dietary antioxidants on the mutagenicity of 7,12-dimethylbenz[a]anthracene and bleomycin in female rats.

Studies on agents that modulate carcinogen-induced genotoxic effects in experimental animals provide end points that can be used for assessing the antimutagenic or anticarcinogenic properties of putative chemopreventive compounds and for predicting their protective efficacy in humans. In this study, we investigated the ability of the dietary antioxidant Vitamins C, E, beta-carotene and the mineral selenium to inhibit the mutant frequency (MF) induced by treatment of rats with 7,12-dimethylbenz[a]anthracene (DMBA), a mammary carcinogen and bleomycin (BLM), an anti-tumor agent that can damage DNA by free radical mechanisms. Both chemicals have been previously shown to be mutagenic in the rat lymphocyte Hprt assay. Adult female Fischer 344 rats were given the antioxidants singly or in a combination 2 weeks prior to mutagen treatment. Antioxidant intake continued for an additional 4 weeks post-mutagen treatment. At sacrifice, spleens were aseptically removed for the isolation of lymphocytes to conduct the mutagenesis assay at the Hprt locus. The DMBA and BLM treatment induced a marked increase in MF, 52.8 x 10(-6) and 19.2 x 10(-6), respectively, over the controls. The MFs seen in the individual antioxidants alone (single or mixture) were relatively similar to the controls, with the exception of Vitamins C and E, that had 1.7- and 1.5-fold increase, respectively. The degree of inhibitory response was dependent on the type of mutagen and the particular antioxidant. BLM/antioxidant combination had inhibitions ranging from 44 to 80%, while DMBA/antioxidant system ranged from 60 to 93%, with Vitamins C and E achieving the highest inhibition in both systems. The mixture displayed low inhibitory responses, 44.6% for BLM/mix and 47% DMBA/mix. On the whole, the results indicate that the dietary constituents tested are antimutagenic; however, because of the gradations seen with the responses, the protective efficacy of these antioxidants may depend on the type of mutagen/carcinogen they encounter. Pending molecular analysis of mitochondrial DNA mutations will also indicate whether there is a shift in the mutational spectra produced by the carcinogens in the presence of antioxidants.

Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats.

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.

Effects of bleomycin on liver antioxidant enzymes and the electron transport system from ad libitum-fed and dietary-restricted female and male Fischer 344 rats.

Dietary restriction (DR) is the only known intervention that delays aging and age-related diseases. Mechanisms proposed to explain this DR effect include a decline in free radical production and an increase in free radical detoxification. In the present study the effect of bleomycin (BLM) as a reactive oxygen species-generating antitumor drug has been evaluated on antioxidant enzymes and the electron transport system in different cellular fractions of liver in female and male Fischer 344 rats. Animals were fed ad libitum (AL) or 60% of the AL intake (DR) and were given a single intraperitoneal injection of 2.5, 5, or 10 mg BLM/kg body wt. After four weeks, BLM significantly increased glutathione peroxidase and lactate dehydrogenase activities in liver cytosol of female AL rats and increased activity even more in male rats. Similar changes were also noted for glutathione reductase and glucose 6-phosphate dehydrogenase activities in BLM-treated AL rats. In liver mitochondria, glutathione peroxidase was increased in female and male AL rats but was increased more in female rats. Drug treatment had no significant effect on these enzyme activities in cytosolic or mitochondrial fractions of DR animals. Profound effects of BLM were noted in activities of complexes I, III, and IV of the electron transport system in AL and DR female and male rats; however, complex II demonstrated no significant diet or treatment effect. Induced antioxidant enzyme activities in BLM-treated AL rats may be a response to excessive free radical generation due to BLM metabolism in AL animals that is mitigated by DR. Furthermore, dysfunction of the electron transport system might suggest its role in a secondary generation of free radicals during BLM metabolism contributing to its toxicity.

Attenuation of bleomycin-induced Hprt mutant frequency in female and male rats by calorie restriction.

Calorie restriction modulates spontaneous and chemically induced tumors and increases maximal life span in experimental animals; however, the mechanism by which calorie restriction exerts its ameliorating effects is not fully elucidated, although reduced levels of reactive oxygen species (ROS) by calorie restriction has generated much interest. In the present study, we have determined whether or not calorie restriction would affect the mutagenic response in rats treated with bleomycin (BLM) a radiomimetic drug that is associated with DNA damage by a free radical mechanism. Fourteen weeks after weaning, the rats were divided into two groups; ad libitum (AL)-fed and 40% calorie restriction. Both AL and calorie-restricted animals were injected with 2.5, 5.0 and 10.0 mg BLM/kg, or with phosphate-buffered saline (PBS), and they were killed 4 weeks post drug treatment. Lymphocytes from the spleens were seeded in 96-well microtiter plates to determine mutant frequency in the hypoxantine guanine phosphoribosyl transferase (Hprt) gene. The mutant frequency in the BLM-treated rats was higher in AL males (P=0.001), and AL females (P=0.0174) than in their calorie-restricted counterparts. The difference in mutagenic response relative to AL males and AL females appeared unrelated to a low percent cloning efficiency seen in the males, since the mean absolute number of Hprt mutant clones was higher in the AL males compared to the females. A reduction in animal weight by calorie restriction was significant in both sexes (P<0.001), but the dose effect appeared non-significant. The results indicate that calorie intake of 60% reduced the mutagenic response of BLM, a compound known to induce oxidative DNA damage, and suggest a possible decrease in ROS as a function of calorie restriction.

Hprt mutant frequency and molecular analysis of Hprt mutations in Fischer 344 rats treated with thiotepa.

Thiotepa is a bifunctional alkylating anticancer drug that is a rodent carcinogen and a suspected human carcinogen. In order to determine the sensitivity of mutant induction in the Hprt lymphocyte assay for detecting tumorigenic doses of thiotepa, Fischer 344 rats were treated for 4 weeks with thiotepa using a procedure adapted from a carcinogenesis protocol. At various times after beginning the treatment regimen, rats were killed and the lymphocyte Hprt assay was performed on splenic lymphocytes isolated from the animals. The 6-thioguanine-resistant T lymphocyte mutant frequency increased with time during the period of thiotepa exposure and declined slightly thereafter. Significant dose-dependent increases in mutant frequency were found using concentrations of thiotepa that eventually result in lymphoproliferative tumors. Hprt mRNA from mutant lymphocytes was reverse transcribed to cDNA, amplified by PCR and examined for mutations by DNA sequencing. This analysis indicated that the major type of point mutation was G:C-->T:A transversion and that 33% of the mutants contained simple or complex frameshifts. Also, a multiplex PCR performed on DNA from mutant clones that were expanded in vitro indicated that 34% of the clones had deletions in the Hprt gene. These results indicate that the induction of lymphocyte Hprt mutants is a sensitive biomarker for the carcinogenicity of thiotepa and that the types of mutations found in the lymphocyte Hprt gene reflect the kinds of DNA damage produced by thiotepa.

Aflatoxin B1-induced Hprt mutations in splenic lymphocytes of Fischer 344 rats. Results of an intermittent feeding trial.

In a previous study, we found an increase in the mutant frequency at the Hypoxanthine phosphoribosyl transferase (Hprt) locus in the splenic lymphocytes of Fischer 344 rats acutely exposed to aflatoxin B1 (AFB1). Because an acute exposure may not reflect the exposure pattern of individuals whose diet may contain AFB1-contaminated foodstuffs, we sought to determine if the feeding regimen affected the induction of Hprt mutations in the rat splenic lymphocyte. Thus, Fischer 344 rats were fed either (A) a control diet, (B) various doses of AFB1 for three four-week periods interspersed with two four-week periods of the control diet, or (C) continuously fed 1.6 ppm of AFB1. Not only was a significant increase in the mutant frequency detected in the lymphocytes of rats fed a dose as low as 0. 01 ppm of AFB1, but the increase in the mutant frequency at the end of the 20-week experimental period was consistent with an accumulation of damage induced by AFB1. These results indicate that the rat lymphocyte/Hprt assay is useful for detecting chronic low level exposures. Further, these data suggest that an intermittent, low-level exposure to AFB1 may present a human health risk.

Hprt mutant frequency and molecular analysis of Hprt mutations in rats treated with mutagenic carcinogens.

Much of the progress in the field of cancer research has come from the increased understanding of the molecular events associated with the initiation and accumulation of mutational events associated with carcinogenesis. Genetic toxicologists have developed a number of in vitro and in vivo non-mammalian and mammalian systems to predict those genetic events required to induce the cancer process. Several model rodent systems have been proposed that have the ability to detect and quantify in vivo somatic mutation in endogenous genes and transgenes and relate the nature of the mutation to the specific type of chemical damage. One such system, the rat lymphocyte hypoxanthine guanine phosphoribosyl transferase (Hprt) assay is described in this review. Data are presented that describe mutant induction and mutational spectra in N-ethyl-N-nitrosourea (ENU), 7,12-dimethylbenzo[a]anthracene (DMBA) and thiotepa (TEPA) treated rats.

Comparison of mutant frequencies and types of mutations induced by thiotepa in the endogenous Hprt gene and transgenic lacI gene of Big Blue rats.

The utility of the lacI transgene of Big Blue rats as a reporter of in vivo mutation was evaluated by comparing the frequency and types of mutations induced by thiotepa in the transgene and the endogenous Hprt gene. Transgenic rats were given i.p. injections of 1.4 mg/kg of thiotepa three times per week over a period of 4 weeks (a total dose of 16.8 mg/kg); 1 week after the last injection, mutation assays were performed on spleen lymphocytes isolated from the animals. Thiotepa treatment increased the lacI mutant frequency from 34.8 +/- 4.1 x 10(-6) in control animals to 140.9 +/- 64.8 x 10(-6) (p = 0.0020) and the Hprt mutant frequency from 3.5 +/- 1.5 x 10(-6) to 41.1 +/- 23.2 x 10(-6) (p = 0.0028). Sequence analysis of lacI mutant DNA and Hprt mutant cDNA produced similar overall mutation patterns: G:C-->T:A transversion was the most common base pair substitution (32% of independent mutations in the lacI gene and 28% of Hprt mutations), and deletions and insertions accounted for 34% of mutations in the lacI gene and 28% in the Hprt gene. The majority of thiotepa-induced base pair substitutions in the Hprt gene occurred with the mutated purine on the non-transcribed DNA strand, while no strand-related bias was found for mutations in the lacI gene. Substitutions at G:C base pairs in the lacI gene, but not in the Hprt gene, were found disproportionately in CpG sites. In addition, multiplex polymerase chain reaction analysis of genomic DNA from the Hprt mutants indicated that 34% had relatively large deletions; none of these deletions was detected by the cDNA analysis. The results indicate that the frequency of thiotepa-induced mutants in Big Blue rats was 2.8-fold greater in the lacI gene than in the Hprt gene. Although the Hprt gene recovered a fraction of large deletions not found among the lacI mutants, the effects of transcription-coupled DNA repair in the Hprt gene and the targeting of base pair substitutions to G:C base pairs in CpG sites may have contributed to the higher mutant frequencies induced by thiotepa in the lacI transgene compared with the Hprt gene.

Comparison of in vivo mutagenesis in the endogenous Hprt gene and the lacI transgene of Big Blue(R) rats treated with 7, 12-dimethylbenz[a]anthracene.

The lacI transgene of Big Blue(R) (BB) rats was evaluated as a reporter of in vivo mutation by comparing mutant frequencies (MFs) in it and in the endogenous Hprt gene. Seven-week old female BB rats were given single doses of 0, 20, 75 and 130 mg/kg of 7, 12-dimethylbenz(a)anthracene (DMBA) by gavage, and Hprt and lacI MFs in splenic lymphocytes were measured over a period of 18 weeks. The Hprt MFs in treated rats increased for 10 weeks and then declined; 130 mg/kg of DMBA produced a maximum Hprt MF of 168+/-11.4x10-6 clonable lymphocytes, while the MF in control rats was 7.4+/-1. 5x10-6. DMBA exposure of generic F344 rats resulted in a similar time-course of mutant induction but produced about 50% higher Hprt MFs with the 75 and 130 mg/kg doses. In contrast, the lacI MFs increased for 6 weeks and then remained relatively constant; 130 mg/kg of DMBA produced a maximum increase in lacI MF of 341+/-83x10-6 PFU compared with 25+/-5x10-6 PFU in control rats. The Hprt mutant frequencies in DMBA-treated BB and F344 rats were significantly increased over control values for every dose-time combination examined, while only the 130 mg/kg dose consistently produced lacI MFs that were significantly above the controls. In addition, the fold-increase in MF for treated vs. control rats was two times higher for the Hprt gene than the lacI gene due to the higher MFs in the lacI gene of control rats. Differences between the lacI and Hprt genes in the kinetics of mutant induction, in the frequency of induced mutants, and in the sensitivity of mutant detection could be explained at least partially by the properties of these two genes.

Comparison of the types of mutations induced by 7,12-dimethylbenz[a]anthracene in the lacI and hprt genes of Big Blue rats.

An important question regarding the use of transgenic reporter genes to detect mutation in rodents is how the types of mutations recovered in transgenes compare with the types of mutations found in endogenous genes. In this study, we examined mutations induced by 7,12-dimethylbenz[a]anthracene in the lacI transgene and the endogenous hprt gene of lymphocytes from Big Blue rats and in the hprt gene of lymphocytes from nontransgenic Fischer 344 rats. The overall mutation profiles found in these genes were remarkably similar: the majority of mutations were base pair substitutions, with the most common mutation being A:T-->T:A transversion. Differences were found for the mutational profiles in the endogenous gene and transgene with respect to the location of the mutations and the orientation of basepair substitutions in the DNA strands. In most cases, these differences could be explained by the nature of the target genes. The results support the use of the lacI transgene for detecting in vivo mutation.

Expansion of rat 6-thioguanine-resistant T-lymphocyte clones by stimulation with ionomycin and a phorbol ester.

Previous molecular analyses of the mutations produced in the rat lymphocyte hprt assay were hindered by difficulties encountered in growing mutant lymphocytes from 6-thioguanine-resistant clones. In this study, we evaluated the ability of the calcium ionophore, ionomycin, and the tumor promotor, phorbol 12-myristate 13-acetate, to stimulate clone expansion. A medium containing these two agents, along with mitogen-free conditioned medium, was found to expand 64% of 276 mutant clones to at least 5 x 10(5) cells in nine days of culture. Some clones were expanded to more than 4 x 10(6) cells. The procedure appears suitable for propagating rat lymphocyte clones for mutation analysis.

Development and utilization of the rat lymphocyte hprt mutation assay.

Much of the recent progress in the field of genetic toxicology has come from an increased understanding of the molecular and cellular biology of the mammalian organism. Most prominent has been the ability to detect and quantify somatic mutation and relate the nature of the mutation to the specific type of chemical damage. Building upon the foundation of the human lymphocyte hypoxanthine guanine phosphoribosyl transferase (hprt) system, and later, the mouse hprt system, methods for the detection and quantification of hprt mutations in rat lymphocytes were developed. These methods are described in this report as is the ongoing validation of the assay. Additionally, the characterization of the recovered mutants and a comparison of the mutation spectrum in the rat lymphocyte system to the spectrum in cancer genes, such as H-ras and p53, and the spectrum in transgenic systems, such as lacI, are included. The development of the rat lymphocyte hprt system and validation of the assay at the molecular level, provide an effective and reliable measure of genetic damage in an in vivo system which is readily comparable to measurement of genetic damage in the human.

Modulation of antioxidant enzymes in bleomycin-treated rats by vitamin C and beta-carotene.

Bleomycin (BLM), an antineoplastic drug, is known to induce DNA strand breaks and is also mutagenic in mammalian cells; however, its mechanism of action is not well understood. It has been proposed that BLM cytotoxicity is mediated through the generation of reactive oxygen species. We have determined the effects of BLM on endogenous hepatic antioxidant enzymes such as glutathione peroxidase (GPx), glutathione reductase, and glucose-6-phosphate dehydrogenase in rats exposed to BLM in conjunction with dietary vitamins, vitamin C and beta-carotene (BC). Male Fischer 344 rats of two different age groups were treated with BLM in the presence or absence of antioxidant vitamins. In control animals, an age-associated decrease in GPx activity was noted (p < 0.05). The decrease in GPx activity observed in BLM-treated old animals given vitamin C was significant (p < 0.05) compared with BLM-treated young animals fed vitamin C. BC moderately induced GPx and glutathione reductase activities in old BLM-treated animals; however, the increase in GPx was statistically significant (p < 0.05) only compared with old controls. A similar increase was noted in the activities of all the enzymes examined in young animals. Our results indicate that BLM exposure was accompanied by alterations in the activities of endogenous antioxidant enzymes, with a profound increase in activities occurring in old animals. In addition, the observed enzyme activities were modulated by antioxidant vitamin administration. The observation that both vitamins displayed differential effects on the enzyme activities also suggests that vitamin C and BC exert their effects by separate mechanisms.

Lymphocyte mutant frequency in relation to DNA adduct formation in rats treated with tumorigenic doses of the mammary gland carcinogen 7,12-dimethylbenz[a]anthracene.

The ability of the rat lymphocyte hprt assay to detect tissue-specific carcinogens was evaluated using 7,12-dimethylbenz[a]anthracene (DMBA) administered under conditions that result in mammary gland tumors. Fifty-day-old female Sprague-Dawley rats were given single doses of 5 and 20 mg/kg DMBA by gavage, and the frequency of 6-thioguanine-resistant (TGr) T-lymphocytes was measured over a period of 21 weeks. A time- and dose-dependent increase in mutant frequency was found, with a maximum frequency found 9-15 weeks after treatment with 20 mg/kg of DMBA. Rats were also dosed with 1, 2.5, 5, 10, 15 and 20 mg/kg of DMBA and assayed for TGr mutant frequency 10 weeks after treatment. A significant linear dose-response was found, with all the DMBA doses resulting in significant increases in mutant frequency. To determine whether or not DMBA-induced mutants in rat lymphocytes reflected the DNA damage in the target tissue, rats were treated with 5 and 20 mg/kg of DMBA and spleen lymphocytes and mammary gland tissue were assayed for DNA adduct formation 1, 3 and 7 days later. A similar pattern of 32P- postlabeled adducts, involving both dG and dA nucleotides, was found in DNA from both the target tissue and the surrogate lymphocytes. Adduct formation was dose responsive in both tissues, with a 2.3- to 4-fold higher concentration in mammary gland as compared with lymphocytes. These results indicate that the rat lymphocyte hprt assay is sensitive to a mammary gland carcinogen and that similar types of DNA adducts are associated with both the lymphocyte mutants and the mammary gland tumors induced by DMBA.

A role for apoptosis in the toxicity and mutagenicity of bleomycin in AHH-1 tk+/- human lymphoblastoid cells.

The chromosomal mutagen, bleomycin, is also noted for its toxic properties, although the mechanism of cell death is not fully understood. In order to determine if cell death occurred by apoptosis or necrosis, AHH-1 tk+/- cells were exposed to bleomycin and the percentage of viable, apoptotic and necrotic cells quantified by flow cytometry. Logistic regression analysis indicated that the primary manner of cell death was through the apoptosis pathways, that apoptosis was delayed, and that apoptosis was accompanied by an arrest in the G2 phase of the cell cycle. Once apoptosis was established as a mechanism for cell death, the efficiency with which these pathways removed damaged cells from the population was evaluated with the use of specific-locus mutation assays (tk and hprt) as indicators of cells with DNA damage that maintained viability and clonogenicity. Linear regression analysis detected a significant, concentration-dependent increase in the numbers of TFTr clones with the slow-growth phenotype. This suggests that a proportion of cells with bleomycin-induced DNA damage did not undergo cell death by apoptosis and that apoptosis, a mechanism for the destruction of damaged cells, is not fully efficient in the AHH-1 tk +/- cell line.

Characterization of rat lymphocyte primary culture for the development of an in-vitro mutagenesis assay: effect of interleukin-2 and 2-mercaptoethanol on the activities of intermediary metabolism enzymes and cell proliferation.

Efficient energy utilization is essential for cell growth; in an attempt to improve the growth conditions of the rat T-lymphocyte culture model for potential use in studying the mutagenic activity of carcinogens in vitro, we have investigated the effects of phytohemagglutinin (PHA), interleukin-2 (IL-2) and 2-mercaptoethanol (2-ME) on the activities of intermediary metabolism enzymes and cell proliferation. Isolated lymphocytes were cultured in the presence and absence of PHA, IL-2, or 2-ME. The intermediary metabolism enzymes investigated were glutamate dehydrogenase, glutamate-pyruvate transaminase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, pyruvate kinase, and fatty acid synthetase (FAS). Measurable activity of all enzymes investigated, except for FAS, was detected in PHA-stimulated cells cultured with IL-2 or 2-ME. The unstimulated lymphocytes had significantly lower enzyme activity than stimulated cells. The combination of all three agents showed increased enzyme activity. This increase in activity brought about by the combination of the three agents was not reproduced by either agent acting alone. In general, the increase in enzyme activity correlated with cell proliferation as measured by [3H]thymidine uptake in PHA-stimulated cultures containing IL-2 and/or 2-ME. The results suggest that the addition of exogenous IL-2 and 2-ME enhances metabolic function and may be beneficial in in vitro culture of rat lymphocytes.