Hypoxanthine-guanine Phosphoribosyl Transferase Gene Locus Research Articles

Alternaria toxins: Altertoxin II is a much stronger mutagen and DNA strand breaking mycotoxin than alternariol and its methyl ether in cultured mammalian cells

Altertoxin II (ATX II) is one of the several mycotoxins produced by Alternaria fungi. It has a perylene quinone structure and is highly mutagenic in Ames Salmonella typhimurium, but its mutagenicity in mammalian cells has not been studied before. Here we report that ATX II is a potent mutagen in cultured Chinese hamster V79 cells, inducing a concentration-dependent increase of mutations at the hypoxanthine guanine phosphoribosyltransferase gene locus at concentrations similar to that of the established mutagen 4-quinoline-N-oxide. Thus, ATX II is at least 50-times more potent as a mutagen than the common Alternaria toxins alternariol (AOH) and alternariol methyl ether (AME). In contrast to AOH and AME, ATX II does not affect the cell cycle of V79 cells. ATX II also causes DNA strand breaks in V79 cells, with a potency again exceeding that of AOH and AME. The high mutagenic and DNA strand breaking activity of ATX II raises the question of whether this Alternaria toxin poses a risk for public health, and warrants studies on the occurrence of ATX II and other perylene quinone-type mycotoxins in food and feed.

Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Mutations in the Asian Population

Mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch– Nyhan syndrome, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior, or HPRT-related gout (Kelley–Seegmiller syndrome). The marked heterogeneity of HPRT deficiency is well known, with more than 300 mutations at the HPRT gene locus having been reported (deletions, insertions, duplications, abnormal splicing, and point mutations at different sites of the coding region from exons 1 to 9). We have identified mutations in Asian families with patients manifesting different clinical phenotypes, including rare cases of female subjects, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse-transcribed mRNA using the polymerase chain reaction technique coupled with direct sequencing. We developed suitable methods to detect the mutations identified from respective families with HPRT deficiency. Then, prenatal genetic diagnoses in HPRT-deficient families were carried out using both mRNA and genomic DNA from chorionic villi or amniotic fluid cells. As shown here in the heterogeneity of HPRT mutations, the spectrum of 70 mutations identified in the Asian population fits the four main conclusions that emerged previously from worldwide analysis.

Acrylamide-induced molecular mutation spectra at HPRT locus in human promyelocytic leukaemia HL-60 and NB4 cell lines

Acrylamide (AA) is a compound widely used in many industries around the world. The recent finding that it is formed naturally in foods by heating raises human health concerns. AA is a proven carcinogen in animals and a probable carcinogen in humans, while its mutagenicity detected using in vitro mammalian gene mutation assays is still inconsistent in different cell systems. In the present study, we investigated the mutagenicity of AA in human promyelocytic leukaemia cells, HL-60 and NB4 cells, by examining the mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus. In a 6-h treatment without the exogenous activation, AA exerted a weak mutagenic effect at the highest concentration used in the study (700 mg/l) in HL-60 cells (P < 0.01) as well as in NB4 cells (P < 0.05). Molecular analysis of AA-induced mutants revealed a different mutation spectrum, when compared to that of spontaneous mutants. The most frequent spontaneous mutations were point mutations, whereas AA-induced mutations were mainly single exon deletions besides point mutations, and an increase in the proportion of partial deletion was associated with the increase of AA treatment. There was no obvious difference in the mutation spectra observed between the HL-60 and NB4 cell lines. These results showed that AA has a weak mutagenic effect at HPRT gene locus in human promyelocytic leukaemia HL-60 and NB4 cell lines and those molecular mutation spectra (single exon deletions and point mutations) may be related to some specific and precise mechanism.

Increased mutant frequencies in the HPRT gene locus of leukemia HL-60 cells treated with succinylacetone

Succinyl acetone (SA) was initially identified in the urine of patients with tyrosinemia type I, an autosomally recessive inherited disease. SA has been used to downregulate the activity of myeloperoxidase (MPO) through its specific inhibition of heme biosynthesis and to investigate the biological properties of MPO in the human myeloid leukemic (HL-60) cell line. The goal of this study is to evaluate the mutagenic potential of SA by determining the frequencies of somatic mutations in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) reporter gene in HL-60 cells following treatment with the chemical. Treatments of HL-60 cells with 500 micromol/L SA for 72 h, a condition generally used to inhibit the MPO activity, resulted in a significantly increased HPRT mutant frequency (HPRT-Mf), compared with the control of untreated cells (47.25 x 10(-6) versus 7.5 x 10(-6), respectively, p <0.01). Treatment of the cells with lower doses of SA also led to an increase in HPRT-Mf but this was significant only with 200 micromol/L (28.67 x 10(-6), p<0.05) and not with doses lower than 100 micromol/L (p0.05), compared with the control of untreated cells (7.5 x 10(-6)). These data show a dose-response increase in HPRT-Mf in HL-60 cells treated with SA, suggesting that this chemical causes mutations in the HPRT locus in these cells either directly or indirectly through its inhibition of the MPO activity.

Cadmium Down-regulates Human OGG1 through Suppression of Sp1 Activity

Cadmium is a well known human and animal carcinogen and is a ubiquitous contaminant in the environment. Although the carcinogenic mechanism of cadmium is a multifactorial process, oxidative DNA damage is believed to be of prime importance. In particular, cadmium suppresses the capacity of cells to repair oxidative DNA damage. In this study, cadmium treatment led to a significant increase in gamma-ray-induced 8-oxoguanine (8-oxoG) formation. Western blotting and semiquantitative reverse transcription-PCR revealed that cadmium treatment caused a decrease in the expression level of human OGG1 (8-oxoguanine-DNA glycosylase-1; hOGG1) in human fibroblast GM00637 and HeLa S3 cells. In addition, the cadmium-mediated decrease in hOGG1 transcription was the result of decreased binding of the transcription factor Sp1 to the hOGG1 promoter. Finally, we show that an increase in the functional hOGG1 expression level could inhibit the cadmium-mediated increase in gamma-ray-induced 8-oxoG accumulation as well as in gamma-radiation-induced mutation frequency at the HPRT (hypoxanthine-guanine phosphoribosyltransferase) gene locus. These results suggest that cadmium attenuates removal of gamma-ray-induced 8-oxoG adducts, which in turn increases the mutation frequency, and that this effect might, at least in part, result from suppression of hOGG1 transcription via inactivation of Sp1 as a result of cadmium treatment.

Effect of dietary intake and lifestyle factors on in vivo mutant frequency at the HPRT gene locus in healthy human subjects

This paper describes the results of a study designed to assess the effects of a variety of dietary and lifestyle factors on background levels of mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyltransferase ( HPRT) gene locus in humans. Eighty-three healthy and free-living subjects (aged 20–80 yr; 61 males and 22 females; mean age of 63.07±14.71 yr) were recruited. Background levels of MF were determined for each subject using a cloning assay. The mean MF/10 6 clonable cells (MF) for the study subjects was 4.63±2.20. An interview-administered questionnaire was completed by each study subject in order to assess details of dietary history, physical activity, health and potential genotoxin exposure history. A 7-day estimated dietary record method with a food frequency questionnaire was used to determine average intakes of energy and macronutrients (including alcohol), and a range of micronutrients (including vitamin and mineral supplement usage). The relationships between individual dietary and lifestyle factors and HPRT MF were investigated by univariate and multivariate analysis (data was adjusted for age, lymphocyte plating efficiency [PE] and energy intake [EI]). Univariate analysis revealed a significant positive correlation between EI and MF and multivariate analysis revealed significant positive correlations between, body mass index (BMI), % energy intake from total carbohydrate, starch, fat and MF. These findings suggest that a reduction in EI may be a useful preventative measure against the onset of carcinogenesis in humans. No correlations were found between alcohol intake and MF or between estimated antioxidant intake and MF. Thus, estimated intakes of antioxidants may not reflect their bioavailability and functional capacity in vivo and it may be more useful to examine actual plasma/cell levels vs. MF to establish if any significant relationship exists.

Increase in Radiation-Induced HPRT Gene Mutation Frequency after Nonthermal Exposure to Nonionizing 60 Hz Electromagnetic Fields

It is widely accepted that moderate levels of nonionizing electric or magnetic fields, for example 50/60 Hz magnetic fields of about 1 mT, are not mutagenic. However, it is not known whether such fields can enhance the action of known mutagens. To explore this question, a stringent experimental protocol, which included blinding and systematic negative controls, was implemented, minimizing the possibility of observer bias or experimental artifacts. As a model system, we chose to measure mutation frequencies induced by 2 Gy gamma rays in the redox-sensitive hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in Chinese hamster ovary cells. We tested whether a 12-h exposure to a 60 Hz sinusoidally oscillating magnetic-flux density (Brms = 0.7 mT) could affect the mutagenic effects of ionizing radiation on the HPRT gene locus. We determined that the magnetic-field exposure induced an approximate 1.8-fold increase in HPRT mutation frequency. Additional experiments at Brms = 0.23 and 0.47 mT revealed that the effect was reduced at lower flux densities. The field exposure did not enhance radiation-induced cytotoxicity or mutation frequencies in cells not exposed to ionizing radiation. These results suggest that moderate-strength, oscillating magnetic fields may act as an enhancer of mutagenesis in mammalian cells.

In vitro investigation of toxaphene genotoxicity in S. typhimurium and Chinese hamster V79 lung fibroblasts

The polychlorinated pesticide toxaphene has been identified as a persistent environmental contaminant and is of particular concern in the Great Lakes and Arctic regions of Canada. Inconsistencies in published in vitro genotoxicology studies have hindered risk assessments of toxaphene exposure. When toxaphene mutagenicity was re-evaluated in the Ames Salmonella/microsome assay at 10–10,000 μg/plate, a dose-dependent increase in His revertants occurred in all five strains of S. typhimurium tested (TA97, TA98, TA100, TA102 and TA104) with higher mutation frequencies observed in the absence of S9 metabolic activation. However, the mutagenic potential of toxaphene was relatively low with concentrations greater than 500 μg/plate required to induce mutation. Toxaphene genotoxicity was also examined in a mammalian system using Chinese hamster V79 lung fibroblasts with metabolic activation provided by human HepG2 hepatoma cells. Genotoxicity of 1–10 μg/ml toxaphene was examined by measuring the frequency of sister chromatid exchange (SCE) and mutation induction at the hypoxanthine guanine phosphoribosyl transferase ( HGPRT) gene locus. Although small increases in SCE were observed at toxic concentrations of toxaphene approaching the LD 50 (10 μg/ml), they were not found to be statistically significant relative to control. Toxaphene was also unable to induce HGPRT mutagenesis at the concentrations tested. These results show that while toxaphene is a weak, direct-acting mutagen in the Ames Salmonella Test, convincing evidence of dose-dependent SCE induction and mutagenicity at the HGPRT gene locus could not be demonstrated in V79 cells.

The genotoxicity of lucidin, a natural component of Rubia tinctorum L., and lucidinethylether, a component of ethanolic Rubia extracts.

The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA-protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3H/M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic.

Mutagenicity of mono-, di- and tri-nitropyrenes in Chinese hamster ovary cells

1-Nitropyrene (1-NP), 1,3-dinitropyrene (1,3-DNP), 1,6-dinitropyrene (1,6-DNP), 1,8-dinitropyrene (1,8-DNP) and 1,3,6-trinitropyrene (1,3,6-TNP) were tested for mutagenicity in cultured Chinese hamster ovary (CHO) cells. Mutation at the hypoxanthine-guanine phosphoribosyl transferase gene locus was quantified. While 1-NP and 1,3-DNP had only marginal direct-acting mutagenicity, 1,6-DNP, 1,8-DNP and 1,3,6-TNP showed definite mutagenicity, with specific mutagenic activities of 8.1, 21 and 54 mutants/10 6 survivors/μg·ml −1 respectively. The mutagenicity of 1-NP increased with increasing concentrations of Aroclor-1254 induced liver homogenate (S9) in the treatment medium. However, S9 at all concentrations tested decreased the mutagenicity of 1,6-DNP and 1,8-DNP. S9 at low concentrations enhanced the mutagenicity of 1,3-DNP and 1,3,6-TNP and that at high concentrations decreased their mutagenicity. The positive mutagenic response of the nitropyrenes suggests that they are potentially carcinogenic, and that further research into their possible human health risk should be performed.

Diesel-exhaust-particle extract enhancement of chemical-induced mutagenesis in cultured Chinese hamster ovary cells: Possible interaction of diesel exhaust with environmental carcinogens

To investigate the possible interaction of diesel exhaust and other environmental carcinogens, the effect of diesel-exhaust-particle extracts on the chemically-induced mutant frequency at the hypoxanthine-guanine phosphoribosyl transferase gene locus in Chinese hamster ovary (CHO) cells was studied. A direct-acting mutagen, N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), and a mutagen that required metabolic activation, benzo[ a]pyrene [B( a)P], were used. 5 organic extracts, each prepared from the exhaust particles of a different diesel passenger car, were studied alone and in combination with the known mutagens. Combined treatments of the cells with an exhaust-particle extract and a mutagen yielded an observed mutant frequency higher than the expected value (the sum of their individual effects). In the absence of exogenous activation, the observed mutant frequency induced by the combined treatment of CHO cells with diesel exhaust extract and MNNG was 1.4–1.9 times the expected value. In the presence of exogenous activation, using B( a)P as mutagen, the observed mutant frequency was 2.8–3.2 times the expected value. This finding suggests the presence of comutagens/cocarcinogens in the diesel-exhaust particle extract.