Evolutionary pattern and process within the Vertigo gouldii (Mollusca: Pulmonata, Pupillidae) group of minute North American land snails.

A phylogenetic analysis of 19 sibling taxa in the Vertigo gouldii group was conducted on 73 individuals sampled across North America using DNA sequence data of the mitochondrial genes cytochrome oxidase subunit 1 (CO1) and 16S ribosomal RNA (16S), and the internal transcribed spacer-2 of the nuclear ribosomal RNA (ITS-2) gene. The results of these analyses were found incongruent with previous taxonomic concepts used to define the V. gouldii group and its composite taxa that were based entirely on conchological features. The mtDNA sequence data suggest that some previous members of the traditional V. gouldii group may be more closely related to V. modesta. They also suggest that V. gouldii may itself consist of seven species-level branches spread across two deeply rooted clades. Revision of geographical distributions on the basis of these analyses suggests that these Vertigo species may commonly possess continental-sized ranges in spite of their minute size and limited active dispersal ability. High levels of sympatry within the group are also confirmed, with up to four species being known to co-occur within single microsites. These data also suggest that rates of diversification have been non-constant. Assuming a 1%/my rate of base pair substitution, a 10-fold diversification pulse is indicated from 6.7-7.0 myBP, which would be co-incident with known mid-late Miocene global climate changes.

Expression of cytochromes P450 and glutathione S-transferases in human prostate, and the potential for activation of heterocyclic amine carcinogens via acetyl-coA-, PAPS- and ATP-dependent pathways.

Dietary factors appear to be involved in the high incidence of prostate cancer in "Westernized" countries, implicating dietary carcinogens such as heterocyclic amines (HAs) in the initiation of prostate carcinogenesis. We examined 24 human prostate samples with respect to their potential for activation and detoxification of HAs and the presence of DNA adducts formed in vivo. Cytochromes P450 1B1, 3A4 and 3A5 were expressed at low levels (<0.1-6.2 pmol/mg microsomal protein). N-Acetyltransferase (NAT) activities, using p-aminobenzoic acid (NAT1) and sulfamethazine (NAT2) as substrates, were <5-5,500 and <5-43 pmol/min/mg cytosolic protein, respectively. Glutathione S-transferases (GSTs) P1, M2 and M3 were expressed at 0.038-1.284, 0.005-0.126 and 0.010-0.270 microg/mg cytosolic protein, respectively; GSTM1 was expressed in all GSTM1-positive samples (0.012-0.291 microg/mg cytosolic protein); and GSTA1 was expressed at low levels (<0.01-0.11 microg/mg cytosolic protein). Binding of N-hydroxy-PhIP to DNA in vitro occurred primarily by an AcCoA-dependent process (<1-54 pmol/mg/DNA), PAPS- and ATP-dependent binding being <1-7 pmol/mg DNA. In vivo, putative PhIP- or 4-aminobiphenyl-DNA adducts were found in 4 samples (0.4-0.8 adducts/10(8) bases); putative hydrophobic adducts were found in 6 samples (8-64 adducts/10(8) bases). Thus, the prostate appears to have low potential for N-hydroxylation of HAs but greater potential for activation of N-hydroxy HAs to genotoxic N-acetoxy esters. The prostate has potential for GSTP1-dependent detoxification of ATP-activated N-hydroxy-PhIP but little potential for detoxification of N-acetoxy-PhIP by GSTA1. However, there were no significant correlations between expression/activities and DNA adducts formed in vitro or in vivo, DNA adducts in vivo possibly reflecting carcinogen exposure.

Human alpha class glutathione S-transferases: genetic polymorphism, expression, and susceptibility to disease.

The human alpha class glutathione S-transferases (GSTs) consist of 5 genes, hGSTA1-hGSTA5, and 7 pseudogenes on chromosome 6p12.1-6p12.2. hGSTA1-hGSTA4 have been well characterized as proteins, but hGSTA5 has not been detected as a gene product. hGSTA1-1 (and to a lesser extent hGSTA2-2) catalyzes the GSH-dependent detoxification of carcinogenic metabolites of environmental pollutants and tobacco smoke (e.g., polycyclic aromatic hydrocarbon diolepoxides) and several alkylating chemotherapeutic agents and has peroxidase activity toward fatty acid hydroperoxides (FA-OOH) and phosphatidyl FA-OOH. hGSTA3-3 has high activity for the GSH-dependent Delta(5)-Delta(4) isomerization of steroids, and hGSTA4-4 has high activity for the GSH conjugation of 4-hydroxynonenal. hGSTA4 is expressed in many tissues; hGSTA1-1 and hGSTA2-2 are expressed at high levels in liver, intestine, kidney, adrenal gland, and testis; and hGSTA3 is expressed in steroidogenic tissues. Functional, allelic, single nucleotide polymorphisms occur in an SP1-binding element of hGSTA1 and in the coding regions of hGSTA2 and hGSTA3. The main effects of these polymorphisms are the low hepatic expression of hGSTA1 in individuals homozygous for hGSTA1*B and the low specific activity of the hGSTA2E-2E variant toward FA-OOH. These properties suggest that alpha class GSTs will be involved in susceptibility to diseases with an environmental component (such as cancer, asthma, and cardiovascular disease) and in response to chemotherapy. Although hGSTM1, hGSTT1, and hGSTP1 have been associated with such diseases (on the basis of genetic polymorphisms as indicators of expression), alpha class GSTs have been little studied in this respect. Nevertheless, hGSTA1*B has been associated with increased susceptibility to colorectal cancer and with increased efficacy of chemotherapy for breast cancer. Methods for identification and quantitation of human alpha class GST protein, mRNA, and genotype are reviewed, and the potential for GST-alpha in plasma to be used as a marker for hepatic expression and induction is discussed.

Potential chemoprotective effects of the coffee components kahweol and cafestol palmitates via modification of hepatic N-acetyltransferase and glutathione S-transferase activities.

Coffee drinking has been associated with reduced incidence of colorectal cancer, possibly via chemoprotection/modification of the metabolism of dietary heterocyclic amine carcinogens such as 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) by kahweol and cafestol palmitates (K/C), two components of unfiltered coffee. Using the PhIP-exposed male Fisher F344 rat as a model, K/C have been shown to reduce colonic PhIP-DNA adducts by > 50%. We have used the male F344 rat to investigate the effects of dietary K/C (0.02-0.2% as a 1:1 mixture) on the metabolism of PhIP by N-acetyltransferase- (NAT), sulfotransferase- (SULT), and glutathione-dependent pathways. K/C decreased hepatic NAT-dependent PhIP activation by up to 80% in a dose-dependent manner. Conversely, hepatic glutathione S-transferase (GST) activity/expression increased, e.g., 3-4 fold toward 1-chloro-2,4-dinitrobenzene (total activity), up to 23-fold toward 4-vinylpyridine (rGSTP1), and approximately 7-fold for rGSTA2 protein. These effects had fully developed after 5 days of the test diet and persisted for at least 5 days after withdrawal of K/C. Hepatic glutathione increased two- to threefold and this increase was more short-lived than other changes. K/C did not modify hepatic SULT activity or colon NAT and GST activities. Benzylisothiocyanate and black tea, which have also been shown to reduce the formation of PhIP-DNA adducts in this model, had little effect on hepatic NAT, SULT, GST, or GSH. In primary culture of rat hepatocytes, both kahweol and cafestol palmitates reduced NAT activity by 80%. In summary, the unique potential of K/C to convert rapid acetylators to a slow acetylator phenotype, accompanied by GST induction, might contribute to chemoprevention against cancers associated with heterocyclic amines.

Lack of association between Caucasian lung cancer risk and O6-methylguanine-DNA methyltransferase-codon 178 genetic polymorphism.

The formation of DNA adducts is thought to be a critical step for the induction of chemically induced cancer. O(6)-Methylguanine-DNA methyltransferase (MGMT) is a ubiquitously expressed enzyme that repairs DNA adducts formed by alkylating carcinogens. Thus, genetic polymorphisms of the MGMT that could result in differences in MGMT activity are potential risk factors for cancer. In the present study, we established a convenient and reliable genotyping method for the MGMT codon 178 polymorphism, a Lys (AAG) to Arg (AGG) substitution, using restriction fragment length polymorphism (RFLP), and studied differences in the distribution of this polymorphism in 92 Caucasian lung cancer patients and 85 controls. Frequencies of the "A" and "G" alleles (MGMT codon 178, AAG and AGG, respectively) were 0.91 and 0.09, respectively. The genetic polymorphism of the MGMT codon 178 was linked with that of the MGMT codon 143 (P < 0.05). The distribution of the MGMT codon 178 genetic polymorphism was not significantly different between lung cancer patients and controls. Thus, our study suggests that the MGMT codon 178 (and possibly 143) polymorphisms do not appear to markedly affect lung cancer risk for this population. In addition, we found an apparent 10bp-deletion in the intron before exon 5 by DNA sequencing. Because this "deletion" was observed in all sequenced samples (N = 20), the previously reported human (Caucasian) MGMT gene sequence should be revised to exclude this 10bp segment.

Detoxification of electrophilic compounds by glutathione S-transferase catalysis: determinants of individual response to chemical carcinogens and chemotherapeutic drugs?

The glutathione S-transferases (GSTs) catalyze the GSH-dependent detoxification of reactive electrophiles such as genotoxic chemical carcinogens and cytotoxic chemotherapeutic agents. Allelic polymorphism in the GSTs has been used to investigate the hypothesis that GSTs are involved in susceptibility to human cancers. Such studies have resulted in low penetrance, high prevalence associations between cancer risk and GST polymorphisms. By examination of interindividual variation of GST expression it becomes clear that GST genotype alone is not an accurate predictor of GST expression. GST expression is tissue specific and interindividual variation of expression is at least 7-fold in normal tissues. Thus, populations of the same genotype are actually heterogeneous as regards expression. Similarly, polymorphisms are not effective in all tissues and GST induction is not independent of genotype. Mechanistic models for chemical aspects of colorectal cancer and chemotherapy for breast cancer demonstrate some of the ways by which such interactions can be studied and the potential for future studies.

Association between a glutathione S-transferase A1 promoter polymorphism and survival after breast cancer treatment.

Glutathione S-transferase (GST) enzymes detoxify chemotherapeutic drugs, and several studies have reported differences in survival for cancer patients who have variant genotypes for GSTP1, GSTM1 or GSTT1 enzymes. A recently described polymorphism alters hepatic expression of GSTA1, a GST with high activity in glutathione conjugation of metabolites of cyclophosphamide (CP). To consider the possible influence of the reduced-expression GSTA1*B allele on cancer patient survival, we have conducted a pilot study of breast cancer patients treated with CP-containing combination chemotherapy. GSTA1 genotype was determined by polymerase chain reaction and restriction fragment length polymorphism. Kaplan-Meier methods and Cox proportional hazards models were used to evaluate survival in relation to genotype. Among 245 subjects, 35% were GSTA1*A/*A, 49% GSTA1*A/*B and 16% GSTA1*B/*B; the genotype distribution did not differ by ethnic group, age or stage at diagnosis. Among patients who had 0 or 1 GSTA1*B allele, the proportion surviving at 5 years was 0.66 (95% CI = 0.59-0.72), whereas for GSTA1*B/*B subjects the proportion was higher, 0.86 (95% CI = 0.67-0.95). Significantly reduced hazard of death was observed for GSTA1*B/*B subjects during the first 5 years after diagnosis, hazard ratio (HR) = 0.3, 95% CI = 0.1-0.8. The association varied with time, with no survival difference observed for subjects who survived beyond 5 years. These results, although based on a small study population, describe an apparent difference in survival after treatment for breast cancer according to GSTA1 genotype. Further studies should consider the possible association between the novel GSTA1*B variant and outcomes of cancer therapy.

Novel markers of susceptibility to carcinogens in diet: associations with colorectal cancer.

Red meats cooked at high temperatures generate mutagenic heterocyclic amines, which undergo metabolic activation by hepatic cytochrome P450 1A2 and N-acetyltransferase-2. A primary detoxification pathway involves glutathione S-transferase A1 (GSTA1), which catalyzes the reduction of the carcinogenic N-acetoxy derivative back to the parent amine. Recently, we described a polymorphism in the GSTA1 proximal promoter; the variant (GSTA1*B) allele significantly lowers enzyme expression. In a case-control study, GSTA1*B/*B genotype was associated with an increased risk of colorectal cancer, particularly among consumers of well-done meat. Dietary nitrosamines, which are bioactivated by CYP2A6, represent another potential etiologic factor for colorectal cancer. CYP2A6 converts the caffeine metabolite 1,7-dimethylxanthine (17X) to 1,7-dimethyluric acid (17U); we investigated CYP2A6 activity using the 17U/17X urinary metabolite ratio from case-control subjects who completed a caffeine phenotype assay. The distribution of CYP2A6 activity was significantly different between CRCa cases and controls, with subjects in the medium and high activity groups having an increased risk (P for trend=0.001). GSTA1 genotype and CYP2A6 phenotype should be evaluated as markers of susceptibility to dietary carcinogens in future studies.

Effects of the ADH3, CYP2E1, and GSTP1 genetic polymorphisms on their expressions in Caucasian lung tissue.

Individual differences in lung cancer susceptibility should be considered for effective lung cancer prevention. We investigated the CYP2E1, ADH3, and GSTP1 genetic polymorphisms that biotransform xenobiotic carcinogens, and variations of their enzyme activity in Caucasian lung tissues (N=28), and found a variant distribution in pulmonary ADH and CYP2E1 activity. The ADH3*1/*1 subjects (N=8) showed significantly higher ADH activity than ADH3*2/*2 (N=3) subjects (P<0.01). On the other hand, we found a 5-fold variation in the pulmonary CYP2E1 activity using a sensitive HLPC/EC based technique. A subject with the CYP2E1-c/t allele showed 2-fold higher CYP2E1 activity than subjects with the c/c allele (N=14). GSTP1 expression comprised 83% of the total pulmonary GSTs. However, neither the GSTP1 polymorphism, nor other lifestyle factors, such as age, gender, smoking status, were found to be associated with pulmonary GST expression. In conclusion, subjects with the ADH3*1 allele showed higher ADH activity and acetaldehyde-DNA adducts in lung than other subjects; thus, the ADH3*1 allele could be considered a risk factor for lung cancer.

Interindividual variation and organ-specific patterns of glutathione S-transferase alpha, mu, and pi expression in gastrointestinal tract mucosa of normal individuals.

Glutathione S-transferase (GST) protein in gastrointestinal (GI) tracts of 16 organ donors, from whom all or substantial portions of the GI tract (stomach-colon) were available, was quantitated by HPLC and examined for interindividual variability/consistency of organ-specific patterns of expression. GSTP1, GSTA1, and GSTA2 were major components, and GSTM1 and GSTM3 were minor components. Consistent patterns of organ-specific expression were evident despite a high degree of interindividual variation of expression. GSTP1 was expressed throughout the GI tract and showed a decrease of expression from stomach to colon. GSTA1 and GSTA2 were expressed at high levels in duodenum and small intestine and expression decreased from proximal to distal small intestine. In contrast, GSTA1 and GSTA2 expression in colon and stomach of all subjects was low, particularly for colon where GSTA1 expression was 20- to 800-fold lower than that in corresponding small intestine. These consistent patterns of expression would suggest that compared to duodenum and small intestine, colon and to a lesser extent stomach always have low potential for GST-dependent detoxification of chemical carcinogens and are therefore at greater risk of genotoxic effects, particularly via substrates that are specific for GSTA1. This may be a factor in the greater susceptibility of stomach and colon to cancers compared to duodenum/small intestine.