Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.     

Relative imbalances in the expression of estrogen-metabolizing enzymes in the breast tissue of women with breast carcinoma

Estrogens are a known risk factor for breast cancer. Studies indicate that initiation of breast cancer may occur by metabolism of estrogens to form abnormally high levels of catechol estrogen-3,4-quinones, which can then react with DNA to form depurinating adducts and, subsequently, induce mutations that lead to cancer. Among the key enzymes metabolizing estrogens are two activating enzymes: cytochrome P450 (CYP)19 (aromatase), which converts androgens to estrogens, and CYP1B1, which converts estrogens predominantly to the 4-catechol estrogens that are further oxidized to catechol estrogen-3,4-quinones. Formation of the quinones is prevented by methylation of the 4-catechol estrogens by the enzyme, catechol-O-methyltransferase (COMT). In addition, catechol estrogen quinones can be reduced back to catechol estrogens by NADPH quinone oxidoreductase 1 (NQO1) and/or are coupled with glutathione, preventing reaction with DNA. Thus, COMT and NQO1 are key deactivating enzymes. In this initial study, we examined whether the expression of these four critical estrogen activating/deactivating enzymes is altered in breast cancer. Control breast tissue was obtained from four women who underwent reduction mammoplasty. Breast tissues from five women with breast carcinoma, who underwent mastectomy, were used as cases. The level of expression of CYP19, CYP1B1, COMT and NQO1 mRNAs was quantified from total RNA using a real time RT-PCR method in an ABI PRISM 7700 sequence detection system. The control breast tissues showed lower expression of the activating enzymes, CYP19 and CYP1B1, and higher expression of the deactivating enzymes, COMT and NQO1, compared to the cases. In the cases, the reverse pattern was observed: greater expression of activating enzymes and lower expression of deactivating enzymes. Thus, in women with breast cancer, estrogen metabolism may be related to altered expression of multiple genes. These unbalances appear to be instrumental in causing excessive formation of catechol estrogen quinones that, by reacting with DNA, initiate the series of events leading to breast cancer.     

IQ and blood lead from 2 to 7 years of age: are the effects in older children the residual of high blood lead concentrations in 2-year-olds?

Increases in peak blood lead concentrations, which occur at 18-30 months of age in the United States, are thought to result in lower IQ scores at 4-6 years of age, when IQ becomes stable and measurable. Data from a prospective study conducted in Boston suggested that blood lead concentrations at 2 years of age were more predictive of cognitive deficits in older children than were later blood lead concentrations or blood lead concentrations measured concurrently with IQ. Therefore, cross-sectional associations between blood lead and IQ in school-age children have been widely interpreted as the residual effects of higher blood lead concentrations at an earlier age or the tendency of less intelligent children to ingest more leaded dust or paint chips, rather than as a causal relationship in older children. Here we analyze data from a clinical trial in which children were treated for elevated blood lead concentrations (20-44 microg/dL) at about 2 years of age and followed until 7 years of age with serial IQ tests and measurements of blood lead. We found that cross-sectional associations increased in strength as the children became older, whereas the relation between baseline blood lead and IQ attenuated. Peak blood lead level thus does not fully account for the observed association in older children between their lower blood lead concentrations and IQ. The effect of concurrent blood level on IQ may therefore be greater than currently believed.     

Real Time-PCR HBV-DNA Analysis: Significance and First Experience in Armed Forces

Background

HBV DNA quantitation is used extensively world wide for the diagnosis and monitoring of treatment of Hepatitis B virus (HBV) infection. However, it has still to be popular in India. The aim of this study was to quantitate HBV – DNA by Real time – PCR method in Hepatitis B and in immuno-compromised patients, to compare the results with HBeAg detection and to monitor the response to therapy of chronic Hepatitis B patients to antivirals.

Methods

Ninety one serum samples of Hepatitis group of patients (all HBsAg positive), 41 samples from immuno-compromised patients (all HBsAg negative) and 49 patients of Chronic Hepatitis B group (all HBsAg positive) were the subjects of this first ever study in Armed Forces. Twenty serum samples from healthy volunteers and non-hepatitis B patients served as negative controls. The amplification detection was carried out in a Rotor-Gene 2000-sequence detector

Results

Amongst Hepatitis B group, 33% (30/91) of the samples were positive for HBV-DNA and 26% (24/91) of samples were positive for HBeAg. In the immuno-compromised group of patients 14.6% (6/11) of samples were positive for HIV-DNA and 9.7% (4/41) were positive for HBeAg. Of the Chronic Hepatitis B patients on treatment, all (100%) were positive by HBV-DNA, whereas 29/49 (59.2%) were positive by HBeAg before treatment. After treatment with antivirals, 06/49 (12.2%) were positive by both tests and 11/49 (22.5%) were positive only by HBV-DNA. 32/49 (65.3%) patients became negative serologically after therapy.

Conclusion

HBeAg status did not necessarily reflect HBV-DNA level in the serum, as 10/91 (11%) in the Hepatitis B group, 2/41 (4.9%) in the immuno compromised group and 20/49 (40.8%) patients in the Chronic Hepatitis B group were positive for HBV-DNA but negative for HBeAg. HBV-DNA was not found to be positive amongst any of the negative controls. Real time – PCR is a sensitive and reproducible assay for HBV-DNA quantitation and may be started in Armed Forces referral centers in the near future.Key Words: Real time – PCR, Chronic Hepatitis B, HBV – DNA, Antivirals     

DDT serum concentration and menstruation among young Chinese women

High DDE and DDT concentrations were found to be associated with shortened menstrual cycle length in Laotian immigrants to the United States. We examined this issue in a sample of young Chinese women. A total of 60 women aged 20-24 years were enrolled in three maternal and child health clinics (20 from urban, 20 from suburban, 20 from rural) in Shanghai, China, and vicinity, in 1998. Of these women, 47 who did not use hormonal contraceptives and had valid menstrual cycle characteristics were included in the analysis for associations among serum DDE and DDT concentration and menstrual cycle length, duration of menses, and heaviness of menstrual flow. In univariate analysis, higher p,p'-DDE concentration was associated with longer menstrual cycle length (0.66 day per 10 microg/L, 95% confidence interval [CI]: 0.21, 1.11 day). With adjustment for age, body mass index, education, occupation, and resident location, the estimate was 0.42 day (95% CI: -0.35, 1.19 day). p,p'-DDE was not associated with duration of menses or heaviness of menstrual flow. Neither p,p'-DDT nor o,p'-DDT were associated with menstrual cycle length, duration of menses or heaviness of menstrual flow. The study largely suggests no association between DDE and DDT concentrations and menstrual cycle characteristics in young Chinese women, though the weak-to-no correlation of DDE with menstrual cycle length merits further study.     

Effect of succimer on growth of preschool children with moderate blood lead levels

Growth deficits associated with lead exposure might be ameliorated by chelation. We examined the effect of succimer on growth in 780 children 12-33 months old who had blood lead levels of 20-44 microg/dL and were randomized to receive up to three 26-day courses of succimer or placebo in a multicenter, double-blind trial. The difference in changes in weight and height between succimer and placebo groups at 1-34 months was calculated by fitting cubic splines. The difference in height change in children on succimer compared with placebo was -0.27 cm [95% confidence interval (95% CI), -0.42 to -0.11] from baseline to 9 months, when 99% of children had completed treatment, and -0.43 cm (95% CI, -0.77 to -0.09) during 34 months of follow-up. Similar differences in weight gain were not statistically significant. Although succimer lowers blood lead in moderately lead-poisoned children, it does not have a beneficial effect on growth and may have an adverse effect.     

Metabolism and DNA binding studies of 4-hydroxyestradiol and estradiol-3,4-quinone in vitro and in female ACI rat mammary gland in vivo

Studies of estrogen metabolism, formation of DNA adducts, carcinogenicity, cell transformation and mutagenicity have led to the hypothesis that reaction of certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, with DNA can generate the critical mutations initiating breast, prostate and other cancers. The endogenous estrogens estrone (E1) and estradiol (E2) are oxidized to catechol estrogens (CE), 2- and 4-hydroxylated estrogens, which can be further oxidized to CE quinones. To determine possible DNA adducts of E1(E2)-3,4-quinones [E1(E2)-3,4-Q], we reported previously that the reaction of E1(E2)-3,4-Q with dG produces the depurinating adduct 4-hydroxyE1(E2)-1-N7Gua [4-OHE1(E2)-1-N7Gua] by 1,4-Michael addition (Stack et al., Chem. Res. Toxicol., 1996, 9, 851). We report here that reaction of E1(E2)-3,4-Q with Ade results in the formation of 4-OHE1(E2)-1-N3Ade by 1,4-Michael addition. The N7Gua and N3Ade depurinating adducts formed both in vitro and in rat mammary gland in vivo were analyzed by HPLC with electrochemical detection and, for some samples, by LC/MS/MS. When E2-3,4-Q was reacted with DNA in vitro, the depurinating adducts 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua, which are rapidly lost from DNA by cleavage of the glycosyl bond, were formed (>99% of the total adducts), as well as traces of stable adducts, which remain in DNA unless removed by repair. Similar results were obtained when 4-OHE2 was oxidized by horseradish peroxidase, lactoperoxidase, tyrosinase or phenobarbital-induced rat liver microsomes in the presence of DNA. When 4-OHE2 or E2-3,4-Q was injected into the mammary glands of female ACI rats in vivo and the mammary tissue was excised 1 h later, the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua constituted >99% of the total adducts formed. In addition, 4-OHE2 conjugates formed by reaction of E2-3,4-Q with glutathione were also detected. These results demonstrate that the 4-CE are metabolized to CE-3,4-Q, which react with DNA to form primarily depurinating adducts. These adducts can generate the critical mutations that initiate cancer (Chakravarti et al., Oncogene, 2001, 20, 7945; Chakravarti et al., Proc. Am. Assoc. Cancer Res., 2003, 44, 180).