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13C NMR data have been correlated to Toxic Equivalency Factors (TEFs) of the 29 PCDDs, PCDFs, or PCBs for which non-zero TEFs have been defined. Such correlations are called quantitative spectrometric data-activity relationship (QSDAR) models. An improved QSDAR model predicted TEFs of 0.037 and 0.004, respectively, for 1,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4,7-pentachlorodibenzo-p-dioxin (PeCDD), both among the 390 congeners for which zero value TEFs are assumed. A QSDAR model of Relative Potency (REP) values estimated the corresponding values as 0.115 and 0.020. Results from both models indicated that these two congeners may exhibit significant dioxin-like toxicity. If other such congeners have non-zero toxicity, TEF-based risk assessments of some dioxin-, furan-, or PCB-contaminated sites or foods may underestimate toxicity. Both models were extensively cross-validated and the TEF model was externally validated. We confirmed the predictions by an independent in vitro method, a luciferase gene expression assay based on mouse liver cells that found REPs of 0.027 and 0.013, respectively, for 1,3,7,8-TCDD and 1,2,3,4,7-PeCDD. The QSDAR-estimated and gene-expression assayed values agreed. The models were used to predict activity for an applicability domain including 108 non-2,3,7,8 dioxin, furan, or PCB congeners and 2,3,7,8-tetrachlorophenothiazine, a dioxin analog proposed as a drug candidate. This study showed that QSDAR prediction followed by a relatively inexpensive in vitro assay could be used to nominate a few candidates among hundreds for further investigation. It suggested that in silico and in vitro nomination protocols may facilitate practical risk assessment when chemical family members exhibit different degrees of toxicity operating via a common mechanism.

This study evaluates the effects of age and chronic dietary restriction (DR) on nonneoplastic diseases in rats that were fed the American Institute of Nutrition (AIN)-93M purified diet. Male Sprague-Dawley (SD) rats were divided into an ad libitum (AL) group and a DR group that was fed the AIN-93M diet with intake reduced by 31%. Nonneoplastic disease profiles were developed to clarify whether the AIN-93M diet fulfills long-term nutritional requirements of rats. Subsets of rats were killed at 58 and 114 weeks of age, and histopathology was performed. At 58 weeks of age, the 2 main types of nonneoplastic diseases in AL rats were liver vacuolization and cardiomyopathy. Dietary restriction reduced the severity and incidence of both lesions. At 114 weeks of age, the most common lesions in AL rats were cardiomyopathy, nephropathy, liver vacuolization, and degeneration with renal failure and genitourinary infections causing the greatest mortality. Dietary restriction reduced the incidence and severity of these lesions. Nonneoplastic diseases accounted for 28.9% and 0.0% of total mortalities in the AL and DR groups, respectively; however, there was a higher incidence of unknown deaths in the DR rats (52.6%) compared to AL rats (28.9%), which may have limited the success of DR to improve survival. Although the AIN-93M diet supported chronic rat growth, alterations in some dietary component concentrations may be required to lower body weight in chronic rodent and human studies. Factors such as diet composition and digestibility may alter nonneoplastic diseases and mortality in rats and humans in a similar fashion.

The primary purpose of this study was to evaluate the effects of age and long-term dietary reduction on neoplastic diseases in rats fed the AIN-93M purified diet. Second, pathologic profiles are critical to comprehensive dietary evaluation. Male Sprague-Dawley rats assigned to 2 groups, ad libitum (AL) and dietary restricted (DR), were fed the AIN-93M (casein protein) diet free choice and reduced in amount by 31%, respectively. At 58 weeks of age, the predominant types of lesions in AL and DR rats were pituitary and skin tumors. At 114 weeks of age, the most common lesions were pituitary, adrenal gland, skin, mammary, brain, and pancreatic tumors and mononuclear cell leukemia. However, DR had no significant effect on these lesions. Primary findings demonstrate that DR significantly reduced the total number of tumors per rat and incidence of benign and primary tumors (all organs) but did not reduce the incidence of malignant tumors (all organs). Dietary restriction increased the percentage of unknown deaths. These results may explain why survival rates for AL and DR rats were not significantly different at 114 weeks (43.3 vs 57.5%, respectively). These findings differ from previous studies using NIH-31 cereal diet (Aging Clin Exp Res 2001;13:263; J Nutr 2002;132:101; Aging Clin Exp Res 2003;16(6):68; Aging Clin Exp Res 2004;16:448) where neoplastic lesions rather than nonneoplastic lesions were linked to a significant increase in survival rate among cohorts of DR-fed rats (J Nutr 2002;132:101). Factors such as diet composition and digestibility, although not independent of body weight, may have contributed to differences in rat mortality and may affect humans in a similar manner.

Consumption of phytoestrogens and mycoestrogens in food products or as dietary supplements is of interest because of both the potential beneficial and adverse effects of these compounds in estrogen-responsive target tissues. Although the hazards of exposure to potent estrogens such as diethylstilbestrol in developing male and female reproductive tracts are well characterized, less is known about the effects of weaker estrogens including phytoestrogens. With some exceptions, ligand binding to the estrogen receptor (ER) predicts uterotrophic activity. Using a well-established and rigorously validated ER-ligand binding assay, we assessed the relative binding affinity (RBA) for 46 chemicals from several chemical structure classes of potential phytoestrogens and mycoestrogens. Although none of the test compounds bound to ER with the affinity of the standard, 17beta-estradiol (E(2)), ER binding was found among all classes of chemical structures (flavones, isoflavones, flavanones, coumarins, chalcones and mycoestrogens). Estrogen receptor relative binding affinities were distributed across a wide range (from approximately 43 to 0.00008; E(2) = 100). These data can be utilized before animal testing to rank order estimates of the potential for in vivo estrogenic activity of a wide range of untested plant chemicals (as well as other chemicals) based on ER binding.

Understanding structural requirements for a chemical to exhibit estrogen receptor (ER) binding has been important in various fields. This knowledge has been directly and indirectly applied to design drugs for human estrogen replacement therapy, and to identify estrogenic endocrine disruptors. This paper reports structure-activity relationships (SARs) based on a total of 230 chemicals, including both natural and xenoestrogens. Activities were generated using a validated ER competitive binding assay, which covers a 10(6)-fold range. This study is focused on identification of structural commonalities among diverse ER ligands. It provides an overall picture of how xenoestrogens structurally resemble endogenous 17beta-estradiol (E(2)) and the synthetic estrogen diethylstilbestrol (DES). On the basis of SAR analysis, five distinguishing criteria were found to be essential for xenoestrogen activity, using E(2) as a template: (1) H-bonding ability of the phenolic ring mimicking the 3-OH, (2) H-bond donor mimicking the17beta-OH and O-O distance between 3- and 17beta-OH, (3) precise steric hydrophobic centers mimicking steric 7alpha- and 11beta-substituents, (4) hydrophobicity, and (5) a ring structure. The 3-position H-bonding ability of phenols is a significant requirement for ER binding. This contributes as both a H-bond donor and acceptor, although predominantly as a donor. However, the 17beta-OH contributes as a H-bond donor only. The precise space (the size and orientation) of steric hydrophobic bulk groups is as important as a 17beta-OH. Where a direct comparison can be made, strong estrogens tend to be more hydrophobic. A rigid ring structure favors ER binding. The knowledge derived from this study is rationalized into a set of hierarchical rules that will be useful in guidance for identification of potential estrogens.

The question of whether caloric restriction (CR) is hormetic is addressed in terms of two common definitions of the term. In terms of the older definition, i.e., a growth-stimulatory effect when lower doses of a compound which resulted in growth inhibition at higher doses, CR is better characterized as a co-hormetic (i.e., a paradigm which at relatively "low doses," in combination with some stimulus, will evince increased growth (proliferation) and at higher "doses" will inhibit this increased proliferation) rather than a hormetic agent. Mechanisms such as cellular selection of cellular subpopulations, increases in receptor efficiency, and preservation of cellular proliferative potential can interact with agents and produce increased growth as long as the CR is not too severe. In terms of a broader definition, i.e., nonmonotonic dose-response behavior of a compound for any adverse response, CR appears to be hormetic, both as a result of body weight (BW) loss and other potential mechanisms. The impact of changes in BW, or frank CR, can be considered a component of every test for hormesis, and is thus capable for interaction with any other agent. The changes that BW loss (or CR) induce are so profound that any aspect of an agent's action - metabolism, pharmacokinetics, pharmacodynamics - can modulate the response of an organism to an agent. Similarly, other effects of a chemical that induce BW loss, e.g., physical activity or temperature dysregulation, can also induce dose-response curves that appear hormetic. The interaction of the hormetic agents of BW loss and CR can influence agent tests. Controlling these factors may make it possible to dissect the key components of a hormetic response. In addition, the effects of CR or BW loss appear to extrapolate well across species [Colman R, Kemnitz JW. Aging experiments using nonhuman primates. In: Yu BP (Ed), Methods in Aging Research. CRC Press, Boca Raton, FL, 1999, pp. 249-267]. Thus there is some reason to believe that these hormetic factors may be important for humans, and may already be a factor for tests of potentially adverse agents already conducted in humans.

We have utilized a validated (standardized) estrogen receptor (ER) competitive-binding assay to determine the ER affinity for a large, structurally diverse group of chemicals. Uteri from ovariectomized Sprague-Dawley rats were the ER source for the competitive-binding assay. Initially, test chemicals were screened at high concentrations to determine whether a chemical competed with [3H]-estradiol for the ER. Test chemicals that exhibited affinity for the ER in the first tier were subsequently assayed using a wide range of concentrations to characterize the binding curve and to determine each chemical's IC50 and relative binding affinity (RBA) values. Overall, we assayed 188 chemicals, covering a 1 x 10(6)-fold range of RBAs from several different chemical or use categories, including steroidal estrogens, synthetic estrogens, antiestrogens, other miscellaneous steroids, alkylphenols, diphenyl derivatives, organochlorines, pesticides, alkylhydroxybenzoate preservatives (parabens), phthalates, benzophenone compounds, and a number of other miscellaneous chemicals. Of the 188 chemicals tested, 100 bound to the ER while 88 were non-binders. Included in the 100 chemicals that bound to the ER were 4-benzyloxyphenol, 2,4-dihydroxybenzophenone, and 2,2'-methylenebis(4-chlorophenol), compounds that have not been shown previously to bind the ER. It was also evident that certain structural features, such as an overall ring structure, were important for ER binding. The current study provides the most structurally diverse ER RBA data set with the widest range of RBA values published to date.

The collaborative Interagency Agreement between the National Center for Toxicological Research (NCTR) and the National Institute on Aging (NIA) was aimed at identifying and validating a panel of biomarkers of aging in rodents in order to rapidly test the efficacy and safety of interventions designed to slow aging. Another aim was to provide a basis for developing biomarkers of aging in humans, using the assumption that biomarkers that were useful across different genotypes and species were sensitive to fundamental processes that would extrapolate to humans. Caloric restriction (CR), the only intervention that consistently extends both mean and maximal life span in a variety of species, was used to provide a model with extended life span. C57BI/6NNia, DBA/2JNia, B6D2F1, and B6C3F1 mice and Brown Norway (BN/RijNia), Fischer (F344/NNia) and Fischer x Brown Norway hybrid (F344 x BN F1) rats were bred and maintained on study. NCTR generated data from over 60,000 individually housed animals of the seven different genotypes and both sexes, approximately half ad libitum (AL) fed, the remainder CR. Approximately half the animals were shipped to offsite NIA investigators internationally, with the majority of the remainder maintained at NCTR until they died. The collaboration supplied a choice of healthy, long-lived rodent models to investigators, while allowing for the development of some of the most definitive information on life span, food consumption, and growth characteristics in these genotypes under diverse feeding paradigms.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to be toxic by inducing oxygen free radicals in the mammalian nervous system, especially in the nigrostriatal dopaminergic system. The present study was designed to compare the toxic effects of MPP+, the active metabolite of MPTP, in MN9D neuronal cells that exhibit relatively low levels of catalase activity, as compared to CHO cells, which exhibit high levels of catalase activity. The survival of the MN9D cells in the presence of 250 microM MPP+ was less than 10%, whereas CHO cells exhibited 70% survival at the same concentration of MPP+. The ED50 values of MPP+ in MN9D and CHO cell lines were 60-600 microM, respectively. MN9D cells contain less catalase, an enzyme believed to be involved in the detoxification of free radicals compared to CHO cells. The catalase activity was 2 Units/mg protein in MN9D cells and 30 U/mg protein in CHO cells. The catalase activity in CHO cells increased with increasing MPP+ concentrations from 100-500 microM, however, it decreased at 1 mM MPP+. In contrast, catalase activity in MN9D remained the same at all MPP+ concentrations. When the CHO cells were pre-treated with 10-25 mM 3-aminotriazole (3-AT), which inhibits catalase activity, and exposed to MPP+ at various concentrations, they became susceptible to MPP+. It is evident from these data that the differential susceptibility to MPP+ in these two cell lines are due to differences in catalase activity. In addition, the inhibition of constituentive catalase activity in CHO cells by 3-AT treatment enhances their susceptibility. In conclusion, the study demonstrates that catalase activity represents an important defence mechanism in MPTP-induced toxicity.

The effect of cadmium ion (Cd) and ascorbic acid (Asc) on the induction of oxidative DNA damage and on the activities of antioxidant enzymes were investigated in human lymphoblastoid cells (AHH-1 TK+/-). Cd at low concentrations of 5-35 microM induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and caused nuclear DNA strand breaks. The formation both of 8-OHdG and of DNA strand breaks was dose-dependent at the low Cd concentration; both parameters were linearly correlated with each other (R = 0.932 and P = 0.0209). 8-OHdG formation by Cd plateaued at a Cd concentration of 50 microM. Asc also induced 8-OHdG formation, but it had no synergistic effect with Cd on the formation of 8-OHdG or DNA strand breaks. Cd at the concentration of 50 microM induced the nuclear activity of the antioxidant enzymes, catalase and superoxide dismutase (SOD). Furthermore, Cd caused a decrease in the concentration of reduced glutathione (GSH) and an increase in concentration of the oxidized form (GSSG). While Asc had no observable effect on SOD activity, it did increase nuclear catalase activity in cells. This effect on catalase was synergistic with that of Cd. The linear correlation between 8-OHdG and DNA strand breaks induced by Cd at the lower Cd concentrations (< or = 50 microM), suggested that the extent of formation of DNA strand breaks induced by Cd may be offset by their induction of the formation of 8-OHdG and antioxidant enzyme activities.

Nickel (Ni) and magnesium (Mg) exert separate and interacting effects on cells: Ni is toxic while Mg enhances the transformation response of transformed cells and protects from heavy metal-induced toxicity. Transformed rat liver epithelial cells were used in the soft agar (SA) assay to measure the effect of Ni and/or Mg on the expression of anchorage independence. Cells were exposed to +/- Ni and +/- Mg in a single passage of growth medium (GM) prior to assay in SA. The cells were then treated with +/- Ni and +/- Mg in the SA resulting in a 4 x 4 treatment matrix yielding 16 Ni/Mg combinations. Nickel was expected to decrease the transformation frequency (TF) and did so in 6 of the 16 cases. Magnesium was expected to enhance the TF independently of Ni; Mg increased TF values in 7 of 16 cases. The Ni-Mg interaction occurred in 11 of 16 cases. In general, Mg and Ni effects were observed more in GM than is SA. It is not evident from this study why the Ni, Mg, and Ni-Mg effects are not observed universally, but it is evident that metal-metal interactions are not simply defined or analyzed in biological systems. A refined factorial design may be useful in further separating such interactions. From the point of view of the implementation of the SA assay, in which test substances are typically dose previous to the implementation of putting the exposed cells in SA, it is clear that assay results can be markedly altered by the presence of the test compound in the soft agar.

The interactive effects of the anti-oxidant ascorbate (Asc) and the metals cadmium (Cd, as CdCl2) or nickel (Ni, as NiCl2) on the in vitro formation of breaks in double-stranded deoxyribonucleic acid (d/s DNA) were determined. Concentrations of 50 microM Cd or 200 microM Ni were dosed for 4 hours in factorial combinations with 500 microM Asc in RPMI 1640 medium (7 percent bovine serum) in which AHH-1 TK+/- cells (a spontaneously transformed human B lymphoblastoid cell line by Gentest Corp.) were replicating. In combination with Asc, Cd caused significant d/s DNA breaks (p < 0.01, n = 5), while Cd in the absence of Asc produced only a slight (but not significantly different) amount of d/s DNA damage when compared to the cells with no Cd added. The Asc alone was not damaging. The Cd caused damage to the d/s DNA only when Asc was present. The percent of d/s DNA remaining following the respective treatments was: +Cd+Asc, 13 +/- 3; +Cd-Asc, 46 +/- 8; -Cd+Asc, 54 +/- 5; -Cd-Asc, 55 +/- 7. Conversely, the presence of Ni resulted in increased amounts (percent) of d/s DNA compared to control values: +Ni+Asc, 63 +/- 5; +Ni-Asc, 58 +/- 5; -Ni+Asc, 52 +/- 1; -Ni-Asc, 51 +/- 4, (p < 0.05, n = 3). The contrasting results between Cd and Ni in the presence of Asc may reside in the point of action; while Cd acts directly on DNA, Ni is reported to act on heterochromatin. Although Asc is a recognized anti-oxidant, its presence in the media mixture potentiated d/s DNA damage from the Cd. This may be caused by a Fenton-type reaction in which an antioxidant in the presence of metal generates hydroxyl radicals and consequently d/s DNA breaks. Oxidative reactions between metals, oxygen, and antioxidants such as Asc may represent an important mechanism of cell death, toxicity, and transformation.

Magnesium, an essential metal that is important in the normal functioning of DNA, has been shown to interact with some of the toxic heavy metals in respect to biochemical and molecular mechanisms and in altering the tumorigenic process. This study examined the influence of magnesium in combination with nickel and cadmium in respect to damage of the DNA molecule. The purpose of this study was to evaluate the influence of magnesium on the amelioration of the toxic metals nickel and cadmium in respect to sustaining DNA damage. Two types of lymphocytes were used, i.e., primary Fischer 344 rat splenocytes and AHH-1 TK+/-, a human B-lymphoblastoid cell line that has been spontaneously transformed. These cells were grown in either a magnesium-free or magnesium-supplemented RPMI 1640 medium that was specifically formulated for this study. A 2 x 2 factorial design was employed with magnesium and either nickel or cadmium serving as the two factors. The experimental groups were as follows: +Mg+Ni, +Mg-Ni, -Mg+Ni, -Mg-Ni, with cadmium alternating for the nickel in the subsequent studies. The nickel or cadmium was added at a concentration of 50 mumol/L. The presence of double-stranded DNA was determined in each of the respective treatment groups with the two types of cell lines. Based on the results of this study, nickel is not directly toxic to DNA, whereas cadmium produces damage directly on the DNA molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

While the life-extending and disease-modulating effects of caloric restriction (CR) are well documented in whole animal studies and in correlative experiments using cells taken from CR animals, very few studies have used cells in culture after their removal from the CR-fed animal. In using this in vivo-->in vitro approach we have attempted to examine the proposition that the effects of CR can be transferred to individual cells by analyzing the cellular functions of proliferation and transformation, the activation of oncogenes, and the methylation of DNA as a function only of diet. Pancreatic acinar cells excised from CR-fed Brown-Norway rats and placed in rich medium showed different responses compared to cells from ad libitum (AL)-fed controls. CR had the effect of slowing growth rate and protecting against spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced transformation over 14 passages of cells in culture. At the molecular level, cells from the CR animals showed reduced c-Ha-ras oncogene expression and mutation as well as reduced mutation of the p53 suppressor gene. CR also increased genomic methylation of ras DNA. We conclude that the effects of CR treatment of the animal are transferred to individual cells and note that these responses (decreased proliferation and transformation; depressed oncogene expression and mutation and decreased suppressor gene mutation; and increased oncogene methylation) are cellular and molecular analogs of in vivo weight loss, life extension, and carcinogenesis modulation, which are hallmarks of CR in the whole animal. The fact that these responses are seen generations after the cells are removed from the CR-treated animal indicates that CR causes a permanent predisposition of pancreatic acinar cells to these modulated responses and shows the value of the in vivo-->in vitro protocol in studies that relate diet to cellular and molecular function.

Pancreatic acinar cells were isolated for culture from a young (Y) and an old (O) Brown-Norway or Fischer 344 rat fed an ad libitum (AL) or calorically restricted (CR) diet. The cells were cultured and cellular growth rates were determined as a function of passage number. An overall increase in cellular growth rate and transformation frequency with age and/or AL diet relative to youth as well as a decrease with CR diet were concordant with reported responses in vivo. Transformation frequency was measured in Brown-Norway cells and followed the same pattern as the growth response: AL/O > AL/Y = CR/Y > CR/O. The cellular model is shown to fit the general multistage requirements of the carcinogenic process as well as general age and diet characteristics of pancreatic cancer. This pancreatic acinar cell age-diet approach may prove to be a valuable tool for determining mechanisms of exocrine pancreatic carcinogenesis as well as other disease states; it may also be of utility in in vitro gerontological nutritional and pharmacological studies since some of the age and diet determinants of biological effects appear to be segregable. Propensity of cells from an old and/or AL diet animal for faster growth and for cellular transformation are programmed into the cells by the time of their excision from the animal (as late as 14 months), indicating a heritable component in the model or a mechanism that is dependent upon elements that control gene expression.

The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epithelial-like rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 x the concentration in the regular RPMI 1640 media (100mg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G1 and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G1 with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G1 nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics.

Previous studies indicate that gentian violet (GV), a triphenylmethane dye used in agriculture and human medicine, is a clastogen in vitro and a carcinogen in chronically exposed mice and rats. Data on its genotoxic activity, however, have been incomplete and partly contradictory. Mutagenesis and DNA damage experiments were conducted to re-evaluate the genotoxic potential of GV in both bacterial and mammalian cell systems. GV was mutagenic in Salmonella typhimurium tester strains TA97 and TA104, but there was little mutagenic activity detected in strains TA98 and TA100. A rat liver homogenate fraction (S9) tended to increase mutagenicity. The major microsomal metabolites of GV, pentamethylpararosaniline and N,N,N',N'-tetramethylpararosaniline were less mutagenic in TA97 and TA104, while N,N,N',N"-tetramethylpararosaniline was a weak mutagen in Salmonella. GV was not mutagenic in Chinese hamster ovary (CHO) cell strain CHO-K1-BH4, and was a questionable mutagen in CHO-AS52 cells. While GV produced DNA damage as measured by sedimentation of nucleotids derived from B6C3F1 mouse lymphocytes treated in vitro, no damage was found in lymphocytes isolated from mice dosed with GV. GV was also a weak producer of gene amplification in an SV40-transformed Chinese hamster cell line. The results indicate that GV is a point mutagen in bacteria; however, since similar exposure conditions produced weak mutagenic activity in mammalian cells, GV may be carcinogenic by virtue of its clastogenic activity.

With acridine orange (AO) and monochromatic 460-nm light, the mutation rate of the wild-type strain of Escherichia coli (WP2) was 3-fold higher than that of the endonuclease-deficient strain WP2 (uvrA). In addition, the mutation rates of the recombination-deficient strains WP10 (recA) and Bs-1 (uvrB lexA) were also about 3-fold less than that of wild-type strain. This observation is in striking contrast to the earlier results with AO and 500-nm light in which strains WP10 and Bs-1 yielded mutation rates that were 12-fold and 5-fold greater, respectively, than the wild-type response. The relatively large decrease in mutation rate when the uvrA endonuclease was absent together with structural considerations in the binding of AO to DNA lead us to propose that the major lesions leading to mutations produced by 460-nm light in the presence of AO may be true DNa single-strand breaks and occur before DNA replication.

Binary mixtures of benzo(a)pyrene (B(a)P) and benzo(e)pyrene (B(e)P) produce synergistic mutagenic (comutagenic) responses in Salmonella typhimurium strain TA98 (a frameshift detector). The optimum enhancement (25 X) was found at B(a)P concentration of 0.3 microgram/plate and B(e)P concentration of 1.5 microgram/plate. The response of strain TA100 (mostly a base-substitution detector) is opposite that of TA98, showing antagonism and additivity in similar concentration ranges.

Hepatocytes prepared from rats pretreated for 5 days with 500 mg/kg Aroclor 1254 were found to be unsuitable for use in a modified Salmonella mutagenicity assay. These hepatocytes exhibited low viability, did not readily attach to plastic culture dishes, and produced mutagenicity responses with benzo[a]pyrene (B[a]P) and 2-aminofluorene (2AF) that were greatly enhanced by the addition of an NADPH-regenerating system (NADPH-RS). Shortening the Aroclor pretreatment time to 1 day resulted in hepatocytes that exhibited high viability and readily attached to plastic culture dishes. These hepatocytes produced higher numbers of revertants when used to assay the mutagenicities of B[a]P and 2AF than were produced using hepatocytes from animals that were pretreated for 5 days. These reversion frequencies were also higher than those produced using uninduced hepatocytes and were much less affected by the addition of NADPH-RS than were the reversions mediated by the 5-day preinduced hepatocytes. Liver homogenate postmitochondrial fractions (S9s), which were prepared from rats pretreated with Aroclor for 1 or 5 days, were nearly equal in their ability to mediate the mutagenicity of B[a]P and 2AF in the Salmonella/microsome reversion assay. Qualitative differences between the S9- and hepatocyte-mediated mutagenicity of 2AF were found, however. These results indicate that employing hepatocytes from rats pretreated with Aroclor for 1 day, rather than 5 days, results in an enzymatically induced, more-intact cell population that is capable of detecting the mutagenicity of B[a]P and 2AF in a modified Salmonella reversion assay.

The effect on growth of E. coli of a series of 3-, 4-, and 5-ring unsubstituted polycyclic aromatic hydrocarbons (PAH) is determined at concentrations of 10-5M, 10-6M, and 10-7M. The angular hydrocarbon configurations of 1,2-benzanthracene, 1,2,5,6-dibenzanthracene (DIBA), and 3,4-benzpyrene (BP) promote growth; tetracene and pyrene have little or no effect on growth while at most concentrations anthracene, phenanthrene chrysene, 1,2,3,4-dibenzanthracene, and pentacene inhibit bacterial growth. No universal pattern of concentration effect is manifest, but based on the results an angular acene configuration is a necessary condition for growth stimulation.

Acridine orange (AO) and methylene blue (MB) in the dark were shown to be weak to moderate mutagens (induction of resistance to T5 phage) in repair-deficient strains of Escherichia coli B/r. However, strain WP2 (wild-type) was not mutated by AO in the dark, in confirmation of earlier data. The presence of 2 microM AO reduced by 41% the spontaneous mutation rate in strain WP2, from 4.1 to 2.4 mutants/10(8) cells/generation. In the polymerase I-deficient strain WP6 (polA1), 2 microM AO increased the mutation rate in the dark 14-fold. We propose that both spontaneous and AO-induced mutagenesis in the absence of light occur at the site of semiconservative DNA replication. If the intercalation mechanism for the effects in the absence of light is valid, the wild-type strain (WP2) may be resistant to frameshift mutagenesis induced by intercalated compounds, while the polymerase I-deficient strain (WP6) may be highly suceptible to the presence of an intercalated dye such as AO at the DNA-replication fork. MB and AO likely act through different mechanisms since MB is only a moderate mutagen in strain WP6 and the other repair-deficient strains tested.

In the presence of acridine orange (AO) and monochromatic 500-nm light, the recombination-deficient strain of Escherichia coli, WP10 (recA), showed a 15-fold increase in mutation rate over the wild-type (WP2) strain. Under the same conditions, strain Bs--1 (uvrB lexA lon) showed a 5-fold increase in mutation rate over strain WP2. In contrast, the endonuclease-deficient, strain, WP2s (uvrA), showed a lower AO-500 nm mutation rate than wild-type. The extremely high mutation rate of the recA strain cannot be due to error-prone inducible SOS repair since the inducible recA + function is absent. Repair of the AO-500 nm-induced lesions is likely due to a recA+-dependent, error-free, recombination process. It is concluded that the high mutation rates with AO-500 nm light obtained in chemostat cultures of recA and lexA strains occur as a consequence of errors during semi-conservative DNA replication in the presence of unrepaired DNA lesions.

Previous studies have shown that 1- and 3-nitrobenzo[a]pyrene (NBaP) were mutagenic in the Salmonella reversion assay without exogenous activation and that 1-, 3- and 6-NBaP were mutagenic in the presence of hepatocytes or liver homogenate (S9). In the present study, 1-, 3- and 6-NBaP were tested for mutagenicity in Chinese hamster ovary (CHO) cells under activation conditions similar to those used in the bacterial studies. None of the NBaPs was mutagenic without exogenous activation and none was mutagenically activated by hepatocytes from unpretreated rats or rats pretreated with Aroclor 1254 or 3-methylcholanthrene. Benzo-[a]pyrene (BaP), the parent compound, induced a strong mutagenic response under all hepatocyte mediation conditions. The NBaPs did produce positive mutagenic responses with S9 activation (3- = 1- greater than 6-NBaP), but these moderate responses were less than those of BaP. The difference between the bacterial and CHO results under the variety of activation conditions suggests the importance of the endogenous metabolism of the target cell as well as the source and the type of exogenous metabolic activation.

The mononitro-substituted isomers of benzo[a]pyrene (B[a]P), 1-, 3- and 6-nitrobenzo[a]pyrene (NB[a]P), are environmental pollutants and are metabolized to mutagens in Salmonella by rat-liver homogenate postmitochondrial supernatant (S9) fractions. In this study, activation of these compounds to mutagens was investigated using the hepatocyte-mediated Salmonella mutagenicity assay. Hepatocytes from rats treated with Aroclor 1254 activated both 3-NB[a]P and 1-NB[a]P to mutagens, while 6-NB[a]P was not mutagenic. The positive mutagenicity responses were functions of both the chemical dose and the hepatocyte concentration. By using a nitroreductase-deficient strain (TA98NR) and a transesterificase-deficient strain (TA98/1,8-DNP6), it was verified that the direct-acting mutagenicities of 1- and 3-NB[a]P primarily were due to metabolic processes involving nitroreduction while the S9- and hepatocyte-mediated mutagenicity responses were also dependent on transesterification. When compared with the mutagenic responses produced with S9, the mutations induced by 1- and 3-NB[a]P in the presence of hepatocytes were relatively more dependent upon nitroreductase metabolism and less on transesterification. Thus, intact hepatocytes were capable of activating 1- and 3-NB[a]P to mutagenic metabolites and some of these metabolites appeared to be different from those produced by S9.

Previous studies showed that binary mixtures of the environmental pollutants 1- and 3-nitrobenzo[a]pyrene produced a synergistic mutational response in the Salmonella reversion assay. Since nitroreduction is believed to mediate the direct-acting mutagenicity of the individual compounds, we have examined the role of nitroreduction in the mutagenicity of mixtures of 1- and 3-nitrobenzo[a]pyrene in the Salmonella plate incorporation assay. While mixtures of 1- and 3-nitrobenzo[a]pyrene induced up to 183% more revertants in strain TA98 than produced by equivalent amounts of the individual compounds, in the nitroreductase-deficient strain TA98NR the same mixtures only induced up to 57% more revertants than the individual compounds. Analysis of mixtures of 1- and 3-nitrosobenzo[a]pyrene (the two-electron reduction products of 1- and 3-nitrobenzo[a]pyrene) for mutation induction in TA98 yielded no evidence of a synergistic effect between the compounds. The mutagenicity of the mixtures was dependent upon the amount of the more mutagenic component. Salmonella cultures were also incubated with mixtures of 1- and 3-nitrobenzo[a]pyrene, as well as with equivalent amounts of the individual compounds. In two experiments, nitroreductive ability, as measured by the amount of 1-nitropyrene metabolized to 1-aminopyrene in 1 hr, was increased 9 to 105% in cultures pretreated with the mixtures as compared with cultures pretreated with the individual compounds. The results of this study support the hypothesis that nitroreduction is a major factor in the synergistic mutational response induced by 1- and 3-nitrobenzo[a]pyrene in Salmonella typhimurium.

The polycyclic aromatic hydrocarbons dibenzo(a,i)pyrene and dibenzo(a,h)pyrene, each of which possesses two bay regions, and their bay-region difluorinated derivatives were tested for mutagenicity for ouabain and 6-thioguanine resistance in Chinese hamster V79 cells. Since V79 cells do not metabolize polycyclic aromatic hydrocarbons, mutagenesis was tested in both the presence and the absence of golden hamster embryo cells capable of metabolizing polycyclic aromatic hydrocarbons. Neither of the dibenzopyrenes nor their fluorinated derivatives were mutagenic in the absence of the golden hamster embryo cells. In the presence of these cells (cell-mediated assay), both dibenzopyrenes were mutagenic, whereas the difluorinated derivatives, 2,10-difluorodibenzo-(a,i)pyrene and 3,10-difluorodibenzo(a,h)pyrene, either were inactive or exhibited (on a dose basis) a weak response. However, the mutagenicity of the dibenzopyrenes was eliminated when they were coincubated with 7,8-benzoflavone, a mixed-function oxidase inhibitor. The results suggest that metabolic oxidation of these polycyclic aromatic hydrocarbons at the bay region (presumably to diol-epoxides) is required for a mutagenic response in the cell-mediated assay.

Photodynamic mutagenesis was studied in chemostat cultures of Escherichia coli B/r (TlR trp) exposed to one of six different acridine dyes or methylene blue. Mutation to phage T5 resistance was induced with a broad-spectrum fluorescent-light source. All of the agents tested were photomutagenic; acridine yellow was the most efficient sensitizer and quinacrine was the least efficient. Quinacrine also was moderately mutagenic in the dark, in contrast to the other agents tested, which were not significantly mutagenic in the dark at the low concentrations tested for photomutagenesis. The mutation rate with acridine orange was directly proportional to both fluence rate and dye concentration over the ranges tested. Photomutation rates with acridine orange, proflavine and methylene blue were independent of growth rate of the chemostat cultures. These results are consistent with photomutagenesis occurring as the result of photochemical damage to DNA-dye complexes, independent of cell expression was approximately 2.5 generations for each of the photomutagens tested. This short expression delay supports an earlier segregational model for expression of phage resistance. The following results suggest that photodynamic mutagenesis is due mainly to intercalated dye molecules: (1) both acridine and 9-aminoacridine are photodynamic mutagens; (2) acridine inhibits photomutagenesis with acridine orange; and (3) neither putrescine or spermine, which bind to DNA without intercalating, inhibited photomutagenesis by acridine orange or proflavine.