William W. Au

A multiplex PCR procedure for polymorphic analysis of GSTM1 and GSTT1 genes in population studies

A deletion polymorphism in glutathione S-transferase theta (GSTT1) gene was recently discovered in humans. Similar to the GSTM1 gene, GSTT1 is also recognized as a risk modifier in exposed populations. To evaluate the role of genetic polymorphism in health effects, the combined genetic polymorphism of different genes should be taken into consideration. In the present study, we have developed a multiplex PCR approach for simultaneous replication of both genes for molecular analysis. The multiplex PCR protocol was validated using donor DNA with different polymorphic combinations for both genes from two different ethnic populations (North Americans and Egyptians). The prevalence of the GSTM1 null genotype was 51% among North Americans and 44% among Egyptians. The prevalence of the GSTT1 null genotype was 15% among North Americans and 14.7% among Egyptians. Combined polymorphism analysis of both genes revealed that 6.3% of North Americans harbor the deleted genotype of both genes compared to 8.8% of the Egyptians. The data indicate that there is no major difference in allelic distribution of both genes between the ethnic populations. The multiplex PCR assay used in this study has the advantage of reducing the time, effort and cost required to carry out such analysis. It will also significantly enhance the ability to use genetic screening techniques as a potential tool for early detection of health outcomes in exposed populations.

Biomarkers in molecular epidemiology studies for health risk prediction

The field of molecular epidemiology is very promising, as sophisticated techniques are being developed to address etiology, genetic susceptibility and mechanisms for induction of disease. The use of biomarkers plays a key role in these investigations because the information can be used to predict the development of disease and to implement disease prevention programs. However, as emphasized by Frederica P. Perera, the field is strewn with studies either that failed to use validated biomarkers or whose designs did not adequately consider the biology of the endpoints, and the availability of validated biomarkers of health risk is still limited. In this review, we have briefly described the usefulness of certain biomarkers for the documentation of exposure and early biological effects, with special concern for the prediction of cancer. An emphasis is placed on understanding the biological and health significance of biomarkers. By building reliable biomarker databases, a promising future is the integration of information from the genome programs to expand the scientific frontiers on etiology, health risk prediction and prevention of environmental disease.

Anti-genotoxicity of galangin as a cancer chemopreventive agent candidate.

Flavonoids are polyphenolic compounds that are present in plants. They have been shown to possess a variety of biological activities at non-toxic concentrations in organisms. Galangin, a member of the flavonol class of flavonoid, is present in high concentrations in medicinal plants (e.g. Alpinia officinarum) and propolis, a natural beehive product. Results from in vitro and in vivo studies indicate that galangin with anti-oxidative and free radical scavenging activities is capable of modulating enzyme activities and suppressing the genotoxicity of chemicals. These activities will be discussed in this review. Based on our review, galangin may be a promising candidate for cancer chemoprevention.

The genotoxic effects of adriamycin in somatic and germinal cells of the mouse

The genotoxic effects of adriamycin on somatic and germinal cells were studied in mice treated with single injections of 3, 12 or 24 mg/kg of the drug. From 1 to 5 days post-injection, chromosome aberrations were observed in bone-marrow cells and in diakineses-metaphase I cells from the testes. The frequency of chromosome breakages peaked at 5 h or 1 day for the bone marrow and at 3 and 5 days for the testis. Univalent formation was increased overall but did not have a dose- and time-dependent relationship. In long-term follow-up studies, adriamycin was found to induce cell killing of germline cells which resulted in a reduction in the numbers of spermatocytes and sperm from mice treated with the higher doses. There was complete absence of gametogenetic elements and, eventually, testicular atrophy occurred. In mice treated with 3 mg/kg, there was gradual recovery of spermatogenesis from 50 days onward. Chromosome breaks and translocations were again observed in the recovering spermatocytes. It was concluded that some of the chromosome aberrations must have been induced in the spermatogonial cells which had survived.

Cytogenetic toxicity of gentian violet and crystal violet on mammalian cells in vitro

The cytogenetic toxicity of gentian violet in Chinese hamster CHO cells in vitro has been studied by analyzing (1) squash preparations from direct fixation for recording mitotic anomalies and (2) air-dried preparations (with colcemid--hypotonic pretreatments) for recording metaphase chromosome aberrations. It was concluded that this compound is a mitotic poison as well as a clastogen in vitro. Its clastogenic property was confirmed in 5 other different mammalian cel types. 10 samples of different gentian violet and crystal violet were surveyed and all were shown to be clastogenic. Unless in vivo studies prove otherwise, gentian violet and crystal violet should be regarded as biohazardous substances.

Use of biomarkers to characterize functions of polymorphic DNA repair genotypes.

Inheritance of variant DNA repair genes is believed to influence individual susceptibility to the development of environmental cancer. However, the validity of the belief is dependent upon understanding the functions of the variant genes. Consequently, a variety of studies have been conducted to investigate the functions of variant DNA repair genes, e.g. using biomarkers. These studies on several representative polymorphic DNA repair genes are reviewed in this report. From a general overview, it appears that the biomarker investigations did not provide consistent observations. However, from a more careful evaluation, it is clear that the inconsistencies are probably caused by the use of populations and biomarkers that are not appropriate for investigating the repair activities of the genes. For example, the use of cigarette smokers and patients may not generate precise information for this type of investigations because these conditions can modify the functions of the investigated genes. Thus, the use of healthy non-smokers would be more appropriate. Other problems with these studies includes the small sample size used and the fact that some of the biomarkers used, such as sister chromatid exchanges, are not appropriate because the mechanisms for formation of the biomarkers and their biological significance are unknown. Nevertheless, the following conclusions can be derived from the review of the various biomarker studies that have been published. XRCC1 194Trp, OGG1 326Cys and APE1 148Glu probably have limited alterations in repair activities compared to the wild-type genotypes. XRCC1 399Gln and XRCC3 241Met are deficient in the repair of X-ray-, but not UV-light-induced chromosome aberrations, therefore the variant genes are defective in base excision repair. XPD 312Asn and XPD 751Gln are deficient in the repair of UV-light- but not X-ray-induced chromosome aberrations, therefore they are defective in nucleotide excision repair.

Chromosome aberrations and response to γ-ray challenge in lymphocytes of workers exposed to 1,3-butadiene

An integrated population monitoring study was initiated to investigate whether occupational exposure to current low levels of butadiene is mutagenic to workers. Ten exposed workers (mean production area concentration of 3.5 ppm) and 10 matched plant controls (mean exposure to 0.03 ppm) were selected and blood samples were collected for our study. The standard cytogenetic assay was used to determine chromosome aberration frequencies. In addition, a challenge assay was used to determine response to gamma-rays as an indication of DNA repair deficiencies. In the latter assay, cells were exposed to gamma-rays at the G1 phase of the cell cycle in vitro and the frequencies of chromosome aberrations in the first post-irradiation metaphase cells were quantitated. Based on results of the cytogenetic assay, the exposed group had a higher frequency of cells with chromosome aberrations and higher chromatid breaks per 100 cells compared with the control. However, the difference was not significant (p > 0.1). With the challenge assay, the exposed group had a higher frequency of aberrant cells (p < 0.04), chromatid breaks (p < 0.05), deletions (p < 0.07), and dicentrics (p < 0.02) than the controls. In addition, the dicentric frequencies from workers were significantly correlated with the presence of a butadiene metabolite [1,2-dihydroxy-4-(N-acetylcysteinyl-S)butane] in urine with a correlation of coefficient of 0.6 (p < 0.01). Two outliers were identified and our interpretation of their responses will be discussed. This study indicates that the workers had exposure-induced mutagenic effects. Together with the observation of gene mutation in a subset of the present population, this study indicates that the current occupational exposure to butadiene may not be safe to workers.

Polymorphism of metabolizing genes and lung cancer histology: Prevalence of CYP2E1 in adenocarcinoma

The relationship between genetic predisposition and development of specific cancers has not been adequately elucidated. In this study, the involvement of three polymorphic genes (CYP2E1, GSTM1, and GSTT1) in the development of different histological types of lung cancer was investigated. DNA was extracted from peripheral blood lymphocytes of lung cancer patients who have been long-term cigarette smokers (n = 52). Allelic variants of CYP2E1 were detected using PCR followed by PstI restriction enzyme digest and RFLP analysis, which detects a specific mutation causing over-expression of the gene. GSTM1 and GSTT1 genotypes were detected using two separate differential PCR methods. Our results indicate a 13.5% allele frequency for the CYP2E1 rare PstI site among the lung cancer patients which represents a 3.4-fold increase over the normal controls (OR = 3.5, 95% CL = 0.65-25.8). A novel observation is that all the patients with this polymorphism had adenocarcinomas only, resulting in a significant association between them (OR = 16.17, 95% CL = 0.95-73, P = 0.02). The frequency of the null GSTM1 gene was 42.3% among the lung cancer patients with no preferential tendency towards developing squamous cell carcinoma versus adenocarcinoma (OR = 1.10, 95% CL = 0.3-4.14, P = 0.5). The GSTT1 gene was absent in 21.1% of the patients with a non-significant tendency towards developing squamous cell carcinoma (OR = 1.23, 95% CL = 0.25-6.1, P = 0.5). Another important observation is the significant predominance of the three predisposing polymorphic alleles among the adenocarcinoma patients (OR = 3.4, 95% CL = 0.78-16.1, P = 0.05) compared with the squamous cell carcinoma patients. The results of this study indicate that the inheritance of several polymorphic metabolizing genes, particularly the CYP2E1 gene, contributes not only to the development of lung cancer but also to the development of specific types of cancer.

Further study of the genetic toxicity of gentian violet

The genetic toxicity of gentian violet was studied with the Ames and the Rosenkranz bacterial assays as well as the cytogenetic assays (Chinese hamster ovary cells in vitro in the presence of rat-liver S-9 fractions, the chicken-embryo and mouse-bone-marrow cells in vivo). Gentian violet was found to be toxic but not mutagenic in the Ames assay. However, it was active in the Rosenkranz assay causing reparable DNA damage. The presence of S-9 in the in vitro cytogenetic assay and in the bacterial assays showed that the activity of gentian violet could be reduced or eliminated. In the in vivo assays, gentian violet was not clastogenic and failed to induce sister-chromatid exchanges. However, gentian violet proved to be highly toxic to growing chick embryos at high dosage and depressed mitotic activities in mouse bone marrow after prolonged treatment. Our study suggested that gentian violet can be inactivated by the liver detoxification system. However, it is potentially hazardous to cells that are exposed to the dye directly (e.g. skin epithelium and cell lining of the gastrointestinal tract).

Biomonitoring using accessible human cells for exposure and health risk assessment

A major goal for genetic toxicologist is to provide precise information on exposure and health risk assessment for effective prevention of health problems. A frequently used approach for population study has been to utilize readily available blood cells (lymphocytes and red blood cells) as sentinel cell types to detect biological effects from exposure and to provide early warning signals for health risk. However, such approach still cannot be used reliably for developing strategies in risk assessment and disease prevention. It is possible that other available cell types which are more representative of the target cells for disease may be used to overcome the deficiency. In this report, the use of non-blood cells for biomonitoring is briefly reviewed. Their usefulness in certain exposure condition is highlighted and their effectiveness in documenting exposure compared with other cell types such as the traditional blood cells is presented. It is obvious that the decision in using these non-blood cells in biomonitoring is based on the exposure condition and the experimental design. Nevertheless, monitoring studies using non-blood cells should be encouraged with emphasis on providing dose-response information, comparative response with other cell types and effectiveness for health risk assessment.