Nutritional folate deficiency augments the in vivo mutagenic and lymphocytotoxic activities of alkylating agents

To investigate the interaction of folate deficiency and alkylating agents in vivo, weanling Fischer 344 rats were maintained for 5 weeks on a folate replete, moderately folate deficient, or a severely folate deficient diet. Mutant frequencies at the HPRT locus in splenic lymphocytes were 1.2 ± 0.6, 1.9 ±1.1, and 6.4 ± 4.0 × 10⁻⁶, respectively (P < 0.01). N-nitroso-N-ethylurea (ENU), 100 mg/kg body weight, was much more mutagenic with progressive folate deficiency (5.0 ± 2.4 vs. 16.2 ± 7.3 vs. 39.2 ± 21.0 × 10⁻⁶), suggesting a synergistic interaction (P ≪ 0.01). Neither moderate nor severe folate deficiency significantly enhanced the mutagenic effects of cyclophosphamide, 50 mg/kgbody weight (18.0 ± 7.9 vs. 6.0 ± 2.8 vs. 28.5 ± 28.2 × 10⁻⁶). The number of cloning cells/spleen were reduced 68% in moderately folate deficient rats and by 87% in severely deficient animals (P < 0.05). The combination of folate deficiency and cyclophosphamide reduced the total number of cloning cells further, but ENU alone, or in combination with folate deficiency, did not. These findings indicate that folate deficiency increases the risk of somatic mutations and is lymphocytotoxic in rats. Folate deficiency enhances the mutagenic but not the lymphotoxic effects of ENU, while it increases the lymphotoxic but not the mutagenic activity of cyclophosphamide. Correction of folate deficiency may decrease the immunologic and genetic damage caused by some alkylating agents. Environ. Mol. Mutagen. 32:33–38, 1998 © 1998 Wiley-Liss, Inc.     

The mutagenic activity of 5-bromo-2′-deoxyuridine (BrdU) in vivo in rats

BrdU tablets were implanted subcutaneously in rats, and BrdU concentrations were determined in the serum. Within 5 hr peak concentrations of 10 μg BrdU/ml blood were reached. The influence of BrdU in vivo on cell cycling, DNA synthesis, spontaneous sister chromatid exchange (SCE) frequencies, and gene-mutation frequencies (6-TG^r) was determined in freshly isolated cells from a subcutaneous granulation tissue. The most significant effect of BrdU in vivo was a doubling of the spontaneous 6-TG^r frequency. In reconstruction experiments in vitro the mutagenic activity of BrdU applied in concentrations found in vivo was 2.5–6-fold higher. With the use of agar-coated tablets, BrdU concentrations in the blood were reduced by half, and no peak concentration was found. The differential staining of chromatids was still sufficient. Since the mutagenic effect of BrdU in vitro was found to be strongly concentration dependent, the use of agar-coated tablets is recommended in experiments in which the compound is used to demonstrate SCE in vivo.     

Mutagenicity and carcinogenicity studies of homemade "rust-proof cutting fluid"

A homemade rust-proof cutting fluid (RPCF) used in China was tested for carcinogenicity by an in vivo chronic experiment and for mutagenicity by the Ames Salmonella microsomal assay. Undiluted and threefold water-diluted fluid were given as drinking water to groups of young adult Wistar rats for 2 years. The treatment induced 11/40 malignant tumors with 9/40 acinar adenocarcinomas of the pancreas in the high-dose group. Simultaneous administration of ascorbic acid dissolved in the undiluted fluid at 2 g acid per 1 g sodium nitrite resulted in 1/40 pancreatic carcinoma. The results of the Ames test showed that the technical RPCF was mutagenic to TA100 with or without metabolic activation. It was concluded that the homemade RPCF, which is comprised of sodium nitrite, triethanolamine, and polyethylene glycol, may form direct-acting mutagen(s) upon storage and form, in vivo, e.g., nitrosamines that caused acinar pancreatic carcinoma in Wistar rats. Simultaneous administration of ascorbic acid is suggested for the protection of workers exposed to the rust-proof cutting fluid.     

Quantitative relationship between ethylated DNA bases and gene mutation at two loci in CHO cells

In a previous study we showed that the formation of O⁶-ethylguanine (O⁶-EtGua) in the DNA of CHO cells in culture correlated with mutations induced by ethylnitrosourea (ENU) and diethylsulfate (DES) at the hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus but not at the Na, K-ATPase locus. This study was extended to another ethylating agent, ethyl methanesulfonate (EMS). DNA adduct formation and induction of mutation at the two gene loci were determined simultaneously in CHO cells after EMS exposure. The extent of ethylation at the N⁷ and O⁶ positions of guanine and at the N³ site of adenine were measured and the possible correlations with 6-thioguanine resistance (6-TG^r) and ouabain resistance (oua^r) mutations were investigated. A good correlation between the levels of ethylation at O⁶ guanine and mutation frequency at hprt gene by all three ethylating agents was observed. In the case of the oua^r locus, the frequency of O⁶-EtGua adducts correlated with mutation induction by EMS and ENU but not by DES. Although both EMS and DES have similar reaction mechanisms, these results highlight differences in their mutational specificity. The comparison of this type of analysis with mutational spectra revealed that correlation studies may be inadequate to analyse multi-component phenomena like mutation formation. © 1993 Wiley-Liss, Inc.     

Evaluation of the utility of popliteal lymph node examination in a cyclophosphamide model of immunotoxicity in the rat

The objective of this study was to characterize the variability of rat lymphoid organ weights and morphology following treatment with a known immunotoxicant, with a focus on the usefulness of evaluating popliteal lymph node weight and histology. Cyclophosphamide was administered to male Sprague-Dawley rats by oral gavage at doses of 2, 7 or 12?mg/kg/day for 10 consecutive days. Left and right popliteal lymph nodes (PLN), spleen and thymus were collected at necropsy, weighed, fixed and processed for histopathology. Femoral bone marrow was also collected, fixed and processed for histology. Organ weight variability was greater for PLN than for either spleen or thymus in control animals. There was a significant but weak correlation between paired left and right PLN weights (p?<?0.005; r²?=?0.2774). Significant treatment-related decreases in lymphoid organ weights were observed in spleen and thymus at?≥?7?mg/kg/day (p?<?0.01), whereas in PLN a significant decrease (p?<?0.05) was noted only at 12?mg/kg/day. The inclusion of PLN did not enhance the sensitivity of detection of systemic treatment-related changes in lymphoid organs in a rat cyclophosphamide model.     

Tissue‐specific and time‐dependent clonal expansion of ENU‐induced mutant cells in gpt delta mice

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N ‐ ethyl‐N‐nitrosourea (ENU) with the persistence of ENU‐induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU‐induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis. Environ. Mol. Mutagen. 58:592–606, 2017. © 2017 Wiley Periodicals, Inc.     

Mutational response at the splenic T-Lymphocyte hprt locus in mice treated as neonates: Contrasting effects of the carcinogens N-ethyl-N-nitrosourea,dimethylnitrosamine, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

The newborn mouse tumorigenicity assay, which involves the treatment of animals during the first two weeks after birth and monitoring tumor induction after a year, has been suggested as a cost- and time-effective alternative to the conventional two-year rodent bioassay. In order to evaluate whether or not lymphocyte hprt mutant induction is an accurate predictor of carcinogenicity in the assay, we determined the frequencies of 6-thioguanine-resistant (TG^r) lymphocytes in the spleens of mice neonatally treated with the carcinogenic mutagens N-ethyl-N-nitrosourea (ENU), dimethylnitrosamine (DMN),and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Male C57BL/6 pups were injected on post-natal days 8 and 15, and the frequency of TG^r T-lymphocytes was measured in groups of three animals, sacrificed periodically up to 31 weeks post-treatment. Compared to background frequencies of 1.1–2.9 × 10⁻⁶, mutant frequencies (MFS) reached 155.1 × 10⁻⁶ following a cumulative dose of 49 mg ENU/kg body weight and 172.3 × 10⁻⁶ following a cumulative dose of 142 mg ENU/kg. These results show that TG^r lymphocyte mutations can be induced and measured in mice treated as neonates and that the induced MFs found for mice treated neonatally with ENU are comparable with frequencies reported for the treatment of adult animals with the same chemical. In contrast, treatment with the promutagenic and procarcinogenic compounds DMN (at a maximum concentration of 10.5 mg/kg) and PhIP (26.2 mg/kg) did not result in an increase in lymphocyte MF, suggesting that reactive metabolites of these compounds may not be reaching cells that are sensitive for mutation fixation. The results indicate that the lymphocyte hprt assay may fail to predict the carcinogenicity of some test chemicals in the neonatal mouse bioassay. Environ. Mol. Mutagen. 31:243–247, 1998 © 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Antimutagenic potency of chlorophyllin in the salmonella assay and its correlation with binding constants of mutagen-inhibitor complexes

Chlorophyllin (CHL) is a water-soluble salt of chlorophyll that exhibits antimutagenic activity in short-term genotoxicity assays and inhibits carcinogen-DNA binding in vivo. The antimutagenic potency of CHL was studied against several structurally related heterocyclic amines using the Salmonella assay. The mutagens included 2-amino-3-methylimidazo[4,5,-f]-quinoline (IQ) and seven related IQ-type compounds, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and three additional non-IQ-type compounds. No relationship was observed between mutagenic potency (revertants/ng mutagen) and antimutagenic potency when expressed in terms of the CHL dose/plateinhibiting mutagenicity by 50 percent (l₅₀). However, a correlation was observed between mutagenic potency and the mole ratio of CHL to mutagen giving 50% inhibition (MR₅₀), with most mutagens requiring several hundredfold to several thousandfold molar excess of CHL for inhibition. In spectrophotometric studies, CHL formed noncovalent molecular complexes with the heterocyclic amines, with binding constants in the range 3–13 x 10³ M^?1. Binding constants were inversely correlated with l₅₀ and MR₅₀ values, i.e., with increasing strength of complex formation less CHL/plate and a lower mole ratio of CHL to mutagen was required to inhibit mutagenicity. The results support an inhibitory mechanism in which chlorophylls operate as “interceptor molecules,” interacting with carcinogens and mutagens directly and limiting their bioavailability. © 1993 Wiley-Liss, Inc.     

Applying the erythrocyte Pig‐a assay concept to rat epididymal sperm for germ cell mutagenicity evaluation

The Pig‐a assay, a recently developed in vivo somatic gene mutation assay, is based on the identification of mutant erythrocytes that have an altered repertoire of glycosylphosphatidylinositol (GPI)‐anchored cell surface markers. We hypothesized that the erythrocyte Pig‐a assay concept could be applied to rat cauda epididymal spermatozoa (sperm) for germ cell mutagenicity evaluation. We used GPI‐anchored CD59 as the Pig‐a mutation marker and examined the frequency of CD59‐negative sperm using flow cytometry. A reconstruction experiment that spiked un‐labeled sperm (mutant–mimic) into labeled sperm at specific ratios yielded good agreement between the detected and expected frequencies of mutant–mimic sperm, demonstrating the analytical ability for CD59‐negative sperm detection. Furthermore, this methodology was assessed in F344/DuCrl rats administered N‐ethyl‐N‐nitrosourea (ENU), a prototypical mutagen, or clofibrate, a lipid‐lowering drug. Rats treated with 1, 10, or 20 mg/kg body weight/day (mkd) ENU via daily oral garage for five consecutive days showed a dose‐dependent increase in the frequency of CD59‐negative sperm on study day 63 (i.e., 58 days after the last ENU dose). This ENU dosing regimen also increased the frequency of CD59‐negative erythrocytes. In rats treated with 300 mkd clofibrate via daily oral garage for consecutive 28 days, no treatment‐related changes were detected in the frequency of CD59‐negative sperm on study day 85 (i.e., 57 days after the last dose) or in the frequency of CD59‐negative erythrocytes on study day 29. In conclusion, these data suggest that the epidiymal sperm Pig‐a assay in rats is a promising method for evaluating germ cell mutagenicity. Environ. Mol. Mutagen. 58:485–493, 2017. © 2017 Wiley Periodicals, Inc.     

Mutagenic potency of chlorofuranones and related compounds in salmonella

One of the mutagenic byproducts associated with chlorinated humic waters and kraft pulp bleaching effluents was recently identified as 3-chloro-4-(dichloromthyl)-5-hydroxy-2(5H)-furanone. This compound and several related chlorofuranones and precursors were synthesized and evaluated for direct-acting mutagenicity in Salmonella typhimurium tester strain TA100. Mutagenicity was greatest for 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, its 5-methoxy derivative, and the precursor in their synthesis, 3-(dichloromethyl)-2,4,4-trichloro-2-butenoic acid. Several of the compounds were tested in the presence of added rat liver homogenate S9 fraction, and in all cases mutagenicity was substantially reduced. An important structural feature which may govern the mutagenic response in these instances appears to be the cis arrangement of CHCl₂ and Cl substituents on a carbon-carbon double bond. These compounds may also be transformed in vitro to the same acyclic chlorine substituted alpha, beta-unsaturated aldehyde derivative, which is proposed to be the agent responsible for the observed mutagenicity.     

Mutagenic synergy between paraoxon and plant-activated m-phenylenediamine or 2-acetoxyacetylaminofluorene

Paraoxon (diethyl-p-nitrophenylphosphate) is the toxic, but non-mutagenic metabolite of the organo-phosphorus ester insecticide parathion. Although this agent has been used as a deacetylase inhibitor in many studies, we discovered a mutagenic synergy when paraoxon was incubated with plant-activated m-phenylenediamine (mPDA) or with direct-acting 2-acetoxyacetylaminofluorene (2AAAF) and S. typhimurium tester strains. Using non-toxic concentrations of plant-activated mPDA and paraoxon a 10-fold increase in the mutant yield of S. typhimurium was observed. The mutagenicity of the plant-activated mPDA product required that O-acetyltransferase (OAT) be expressed by the S. typhimurium tester strain. However, the paraoxon-dependent mutagenic synergy was observed using the direct-acting arylamine metabolite, 2AAAF, with strains YG1024, TA98 and TA98/1,8-DNP₆ regardless of their OAT activity. This mutagenic synergy is dependent upon the presence of an activated acetylated form of the arylamine. The data presented here demonstrate that this mutagenic synergy is limited to paraoxon and not to the parent compound (parathion) or to a major metabolite of parathion (p-nitrophenol). © 1996 Wiley-Liss, Inc.     

The following questions were submitted to each participant after the workshop

A compound's mutagenicity in different Salmonella tester strains can suggest its mechanism of reaction with DNA. Clear confirmation of such a mechanism, however, requires a direct test of the compound's reaction with DNA, often relying on specific in vitro studies. We report the use of a rapid in vitro test designed to measure DNA unwinding, a characteristic of DNA intercalators and many frameshift mutagens. CGS 20928A, an adenosine antagonist, produced a significant (<2-fold) increase in revertants only for Salmonella tester strain TA1537, and only without metabolic activation. These data indicated that the compound was a direct acting frameshift mutagen and possibly intercalated into DNA. Our DNA unwinding assay indicated that at concentrations of <0.1 mM CGS 20928A behaved like known intercalating compounds in that it unwound DNA. These concentrations of compound are comparable to those found mutagenic to TA1537. By comparison, the frameshift mutagen and known intercalating compound 9-aminoacridine unwound DNA in this assay in a concentration dependent fashion between 6–12 μM. ICR-191, another acridine frameshift mutagen, also unwound DNA. A compound structurally related to CGS 20928A, which was not mutagenic in Salmonella tester strains, did not produce any DNA unwinding even at 10 mM. Because the assay uses microgram quantities of material, it should be ideal for screening small amounts of congeneric series suspected of frameshift mutagenicity. © 1993 Wiley-Liss, Inc.     

Differential induction of sister chromatid exchanges by indirect-acting mutagen-carcinogens at early and late stages of embryonic development

To examine the development of drug-metabolizing enzyme systems in the early chick embryo, the procarcinogens, aflatoxin B₁ (AF-B₁) and 2-acetylamino-fluorene (2-AAF), and the direct-acting carcinogen, ethyl methanesulfonate (EMS) (positive control), were given to embryos; the sister-chromatid-exchange (SCE) technique was used as an indicator of conversion to active mutagenic metabolities. Chick embryos at two stages of incubation (3-day and 6-day) were exposed to the same graded series of dosages of the compounds for a period of 22 hours. All three mutagens increased the frequency of SCE above the control rate of 1.8 SCEs/cell. While a dose-dependent increase in SCE was obtained for both procarcinogens at each age, the mean SCE frequency was significantly higher in the 6-day embryos for each dosage given. In contrast, the direct-acting mutagen, EMS, gave a reduced level of SCEs at the older age. These results suggest that the ability of early chick embryos to activate promutagens to forms capable of inducing SCE increases as development advances from three to six days of incubation (DI). In the 6-day embryo, the metabolic conversion is enhanced, resulting in a significant increase in the mutagenicity of the test chemicals.     

Transplacental mutagenicity of N-ethyl-N-nitrosourea at the hprt locus in T-lymphocytes of exposed B6C3F1 mice

Previous studies have compared age-related differences in total mutagenic burden in mice of differing age (preweanling, weanling, or young adult) after single intraperitoneal (i.p.) injections of ethylnitrosourea (ENU). The purpose of the present investigation was to determine the effects of time elapsed since treatment on the frequency of hprt mutant T-cells (Mf) from mice treated transplacentally with single acute vs. multiple split doses of ENU. To this end, pregnant C57BL/6 mice (n = 13-16/group), which had been bred to C3H males, were given i.p. injections of 40 mg ENU/kg bw in a single dose on day 18 of gestation, in a split dose of 6 mg ENU/kg bw on days 12 through 18 of gestation, or DMSO vehicle alone. Groups of pups were necropsied on days 10, 13, 15 (single dose only), 17, 20, 40, and 70 postpartum for T-cell isolations and hprt Mf measurements using the T-cell cloning assay. The time required to reach maximum Mfs in T-cells isolated from thymus of transplacentally treated animals was 2 weeks, the same time span as previously observed after ENU treatment of adult, weanling, and preweanling mice. Mfs in T-cells isolated from spleens of control animals averaged 2.1 +/- 0.3 (SE) x 10(-6). In spleens of mice treated transplacentally with ENU in a single dose, Mfs reached a maximum at 15 days postpartum [84.7 +/- 15.8 (SE) x 10(-6)] and decreased to lower but still elevated levels at 40 days postpartum. In spleens of mice treated transplacentally with ENU in a split dose, Mfs reached a maximum at 13 days postpartum [74.0 +/- 16.3 (SE) x 10(-6)] and decreased to background levels at 40 days postpartum. The areas under the curves describing the change in hprt Mfs over time for ENU-treated vs. control mice estimate the mutagenic potency for transplacental single- and split-dose exposures to be 1.9 and 0.8 x 10(3), respectively. Comparison of the mutagenic potency estimates for mice exposed to ENU in utero to 4-week-old mice given a similar dose of the same lot number of ENU indicates that the mouse is more susceptible to ENU-induced mutagenesis during fetal life.     

Cyclopenta[cd]fluoranthene and its precursors in combustion exhausts: a survey of their bacterial mutagenic activity

Cyclopenta[cd]fluoranthene (1) and 3-ethynylfluoranthene (2) have both recently been identified in combustion exhausts. In this study, their mutagenic activities were compared to that of fluoranthene (3), one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in combustion exhausts, in the Salmonella/microsome reversion assay (Ames assay) using S. typhimurium strain TA98. The mutagenicity of 1 was modest in comparison to other active cyclopenta PAHs. Unexpectedly, 2 was mutagenic both with and without exogenous metabolic activation (rat liver S9). Furthermore, cyclopenta[cd]fluoranthene-3,4-epoxide (6) was synthesized in order to evaluate its role as the ultimate mutagenic active form of 1. The epoxide 6 was a direct-acting mutagen. In addition, a pyrolysate containing a mixture of 1 (85%), 2 (2%), and 3 (13%) obtained by flash vacuum thermolysis of 3-(1-chloroethenyl)fluoranthene (2a) at 1,050 degrees C was also mutagenic, but a significant mutagenic response was detected only in the presence of S9 activation. The results of this study indicate that 1 and 2 can contribute to the mutagenic activity of combustion exhausts.     

Diet‐induced obesity increases the frequency of Pig‐a mutant erythrocytes in male C57BL/6J mice

Obesity increases the risk of a number of chronic diseases in humans including several cancers. Biological mechanisms responsible for such increased risks are not well understood at present. Increases in systemic inflammation and oxidative stress, endogenous production of mutagenic metabolites, altered signaling in proliferative pathways, and increased sensitivity to exogenous mutagens and carcinogens are some of the potential contributing factors. We hypothesize that obesity creates an endogenously mutagenic environment in addition to increasing the sensitivity to environmental mutagens. To test this hypothesis, we examined two in vivo genotoxicity endpoints. Pig‐a mutant frequencies and micronucleus frequencies were determined in blood cells in two independent experiments in 30‐week old male mice reared on either a high‐fat diet (60% calories from fat) that exhibit an obese phenotype or a normal‐fat diet (10% calories from fat) that do not exhibit an obese phenotype. Mice were assayed again at 52 weeks of age in one of the experiments. N‐ethyl‐N‐nitrosourea (ENU) was used as a positive mutation control in one experiment. ENU induced a robust Pig‐a mutant and micronucleus response in both phenotypes. Obese, otherwise untreated mice, did not differ from non‐obese mice with respect to Pig‐a mutant frequencies in reticulocytes or micronucleus frequencies. However, such mice, had significantly higher and sustained Pig‐a mutant frequencies (increased 2.5‐3.7‐fold, p < 0.02) in erythrocytes as compared to non‐obese mice (based on measurements collected at 30 weeks or 30 and 52 weeks of age). This suggests that obesity, in the absence of exposure to an exogenous mutagen, is itself mutagenic. Environ. Mol. Mutagen. 57:668–677, 2016. © 2016 Wiley Periodicals, Inc.     

In vitro enhancement of the mutagenicity of 4-nitro-o-phenylenediamine by plant S-9

The direct-acting mutagen 4-nitro-o-phenylenediamine (NOP) was activated in vitro by pea or tobacco S-9 into a more potent mutagen in Salmonella typhimurium strain TA98. NOP mutagenicity was not altered by Aroclor 1254-induced rat liver S-9. The plant S-9 activation of NOP was heat-sensitive but was not NADPH-dependent, did not involve superoxide radicals, and was not inhibited by CO. A direct relationship between plant peroxidase and NOP activation was established. Several purified peroxidases including horseradish peroxidase, chloroperoxidase, and lactoperoxidase also activated NOP. The perodoxidative process was not H₂O₂-dependent but was partially inhibited by a peroxidase inhibitor.     

Structural basis of the mutagenicity in bacteria of nitrated naphthalene and derivatives

While 2-nitronaphthalene was a weak direct-acting base-substitution mutagen (1.4 revertants/nanomole) for Salmonella typhimurium, the analogous nitronaphthalic acid anhydride and imides were moderate frameshift mutagens (～20 rev/nanomole in strain TA98). Although imide derivatives are efficient DNA intercalators, mutagenicity data indicate that the bulk of the frameshift activity is derived from adduct formation between hydroxylamine intermediates and DNA. The low level of frameshift activity (～8% of total) resulting from simple intercalation (measured in strain TA1537) is not dependent upon reduction of the nitro function. Evidence is presented that suggests that the reduction of the nitro function to the corresponding hydroylamine might not involve a free nitroso intermediate. The introduction of a second nitrofunction into nitronaphthalenes results in great positional effects of the various isomers on mutagenic activity and specificity.     

CD4−8− T-cells increase in MRL/lpr mice treated with thymic factors

The in vivo effect of thymic factors on immature lymphocytes was analysed in MRL/lpr mice. This strain carries a genetic defect that causes during their life cycle a block of T-cell differentiation and abnormal proliferation of CD4⁻8⁻ (double-negative, DN) T-lymphocytes. In vivo administration of four preparations of thymic factors, thymopentin (TP-1), thymopoietin (TP-5), thymolymphotropin (TLT), and thymomodulin (TMD) into young (2-month-old) MRL/lpr mice induced a significant increase of DN T-cells both in the thymus and in the peripheral lymph nodes, with a concomitant decrease of double-positive (DP) T-cells in the thymus and of single-positive (SP) T-cells in the lymph nodes. The level of DNA fragmentation measured as propidium iodide fluorescence was increased in the thymus population of young mice and in the lymph node population of old mice treated with TLT. SCID mice transplanted with lymph node cells from MRL/lpr donors (MRL→SCID) developed graft versus host (GvH) reaction due to the activation of MRL CD8⁺ alloreactive T-cells. This model was used to analyse the effect of TMD/TLT in vivo on MRL cell proliferation and expansion; in fact, spleen cells from MRL→SCID mice after treatment with TMD/TLT showed an increased cell proliferation, and an expansion of DN T-cells with a concomitant decrease of SP cells (both CD4⁺ and CD8⁺ cells). Decreased SP cell numbers in this context could explain why TMD/TLT treatment of SCID mice engrafted with MRL cells increased their survival compared to untreated MRL→SCID mice.     

Effects of tea and chlorophyllin on the mutagenicity of N-hydroxy-IQ: Studies of enzyme inhibition,molecular complex formation,and degradation/scavenging of the active metabolites

Green tea and black tea inhibit the formation of carcinogen-DNA adducts and colonic aberrant crypts in rats given 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ), a mutagen from cooked meat. The Salmonella mutagenicity assay was used in the present study to test individual constituents of tea as inhibitors of 2-hydroxyamino-3-methylimidazo[4, 5-f]quinoline (N-hydroxy-IQ), a direct-acting metabolite of IQ. Testing of pure compounds at doses relevant to their levels in tea identified epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG) as the primary antimutagens. Studies of the inhibitory mechanisms established that the rate of degradation of N-hydroxy-IQ under aqueous conditions was not increased significantly in the presence of tea, in contrast to the results obtained with the complexing agent chlorophyllin (CHL), which rapidly degraded the mutagen. Interaction between N-hydroxy-IQ and several tea constituents was detected in spectrophotometric studies, but the binding constants were only on the order of 1 × 10³ M⁻¹, suggesting that mechanisms other than complex formation might prevail under the conditions of the Salmonella assay. Comparison of the results in two different strains of Salmonella typhimurium, TA98 and TA98/1,8-DNP₆, indicated that the antimutagenic activity of EGCG was dependent, at least in part, on a functional O-acetyltransferase activity in the bacteria. These studies suggest that tea constituents inhibit the enzyme(s) which generate the aryl nitrenium ion and directly scavenge the reactive electrophile, whereas CHL complexes with heterocyclic amines and facilitates the degradation of active metabolites. Environ. Mol. Mutagen. 30:468–474, 1997 © 1997 Wiley-Liss, Inc.