Spectrum of point mutations in the coding region of the hypoxanthine- guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes in vivo

The hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in 6- thioguanine (TG) resistant T-lymphocytes is a useful target for the study of somatic in vivo mutagenesis, since it provides information about a broad spectrum of mutation. Mutations in the hprt coding region were studied in 124 TG-resistant T-cell clones from 38 healthy, non- smoking male donors from a previously studied population of bus maintenance workers, fine-mechanics and laboratory personnel. Their mean age was 43 years (range 23-64) and their hprt mutant frequency was 9.3 +/- 5.2 x 10(-6) (mean +/- SD, range 1.4-22.6 x 10(-6)). Sequence analysis of hprt cDNA identified 115 unique mutations; 76% were simple base substitutions, 10% were +/-1 bp frameshifts, and 10% were small deletions within exons (3-52 bp). In addition, two tandem base substitutions and one complex mutation were observed. Simple base substitutions were observed at 55 (20%) of 281 sites known to be mutable in the hprt coding sequence. The distribution of these mutations was significantly different than would be expected based upon a Poisson distribution (P < 0.0001), suggesting the existence of 'hotspots'. All of the 87 simple base substitutions occurred at known mutable sites, but eight were substitutions of a kind that have not previously been reported at these sites. The most frequently mutated sites were cDNA positions 197 and 146, with six and five independent mutations respectively. Four mutations were observed at position 131, and three each at positions 143, 208, 508 and 617. Transitions (52%) were slightly more frequent than tranversions (48%), and mutations at GC base pairs (56%) more common than mutations at AT base pairs (44%). GC > AT was the most common type of base pair substitution (37%). The majority of the mutations at GC base pairs (78%) occurred at sites with G in the non-transcribed strand. All but one of eight mutations at CpG- sites were of the kind expected from deamination of methylated cytosine. Deletion of a single base pair (-1 frameshift) was three times more frequent than insertion of a single bp (+1 frameshift). Almost half (6/13) of the small (3-52 bp) deletions within the coding sequence clustered in the 5' end of exon 2. Short repeats and other sequence motifs that have been associated with replication error were found in the flanking regions of most of the frameshifts and small deletions. However, several differences in the local sequence context between +/-1 frameshift and deletion mutations were also noticed. The present results identify positions 197, 146 and possibly 131 as hotspots for base substitution mutations, and confirm previously reported hotspots at positions 197, 508 and 617. In addition, the earlier notion of a deletion hotspot in the 5'end of exon 2 was confirmed. The observations of these mutational cluster regions in different human populations suggest that they are due to endogeneous mechanisms of mutagenesis, or to ubiquitous environmental influences. The emerging background spectrum of somatic in vivo mutation in the human hprt gene provides a useful basis for comparisons with radiation or chemically induced mutational spectra, as well as with gene mutations in human tumors.      

Hemoglobin and albumin adducts of benzene oxide among workers exposed to high levels of benzene

Benzene oxide (BO) reacts with cysteinyl residues in hemoglobin (Hb) and albumin (Alb) to form protein adducts (BO-Hb and BO-Alb), which are presumed to be specific biomarkers of exposure to benzene. We analyzed BO-Hb in 43 exposed workers and 42 unexposed controls, and BO-Alb in a subsample consisting of 19 workers and 19 controls from Shanghai, China, as part of a larger cross-sectional study of benzene biomarkers. The adducts were analyzed by gas chromatography-mass spectrometry following reaction of the protein with trifluoroacetic anhydride and methanesulfonic acid. When subjects were divided into controls (n = 42) and workers exposed to < or =31 (n = 21) and >31 p.p.m. (n = 22) benzene, median BO-Hb levels were 32.0, 46.7 and 129 pmol/g globin, respectively (correlation with exposure: Spearman r = 0.67, P < 0.0001). To our knowledge, these results represent the first observation in humans that BO-Hb levels are significantly correlated with benzene exposure. Median BO-Alb levels in these 3 groups were 103 (n = 19), 351 (n = 7) and 2010 (n = 12) pmol/g Alb, respectively, also reflecting a significant correlation with exposure (Spearman r = 0.90, P < 0.0001). The blood dose of BO predicted from both Hb and Alb adducts was very similar. These results clearly affirm the use of both Hb and Alb adducts of BO as biomarkers of exposure to high levels of benzene. As part of our investigation of the background levels of BO-Hb and BO-Alb found in unexposed persons, we analyzed recombinant human Hb and Alb for BO adducts. Significant levels of both BO-Hb (19.7 pmol/g) and BO-Alb (41.9 pmol/g) were detected, suggesting that portions of the observed background adducts reflect an artifact of the assay, while other portions are indicative of either unknown exposures or endogenous production of adducts.      

Effect of chronic alcohol consumption on plasma lipid, vitamins A, and E in Korean alcoholics

This human study was conducted to evaluate the effect of chronic alcohol consumption on plasma concentrations of lipid and the antioxidative system in 44 Korean alcoholics and 45 age-, sex-, and nationality-matched nonalcoholic subjects. Plasma triacylglycerols and atherogenic index were higher in alcoholics than in control subjects. Plasma total cholesterol was not different among groups, but plasma high-density lipoprotein cholesterol was lower in alcoholics. There were positive correlations between ethanol consumption and plasma lipid peroxide and atherogenic index in all subjects; there were negative correlations between ethanol consumption and plasma high-density lipoprotein cholesterol in all subjects. There were no significant differences between alcoholics and control subjects in plasma concentrations of α-tocopherol, although plasma α-tocopherol/lipid tended to be lower in alcoholics. Plasma retinol was lower in alcoholics. These results suggest that chronic ethanol consumption can contribute to increased risk for vascular diseases in Korean alcoholics.     

Circulating serum xenoestrogens and mammographic breast density

Introduction

Humans are widely exposed to estrogenically active phthalates, parabens, and phenols, raising concerns about potential effects on breast tissue and breast cancer risk. We sought to determine the association of circulating serum levels of these chemicals (reflecting recent exposure) with mammographic breast density (a marker of breast cancer risk).

Methods

We recruited postmenopausal women aged 55 to 70 years from mammography clinics in Madison, Wisconsin (N = 264). Subjects completed a questionnaire and provided a blood sample that was analyzed for mono-ethyl phthalate, mono-butyl phthalate, mono-benzyl phthalate, butyl paraben, propyl paraben, octylphenol, nonylphenol, and bisphenol A (BPA). Percentage breast density was measured from mammograms by using a computer-assisted thresholding method.

Results

Serum BPA was positively associated with mammographic breast density after adjusting for age, body mass index, and other potentially confounding factors. Mean percentage density was 12.6% (95% confidence interval (CI), 11.4 to 14.0) among the 193 women with nondetectable BPA levels, 13.7% (95% CI, 10.7 to 17.1) among the 35 women with detectable levels below the median (<0.55 ng/ml), and 17.6% (95% CI, 14.1 to 21.5) among the 34 women with detectable levels above the median (>0.55 ng/ml; P_trend = 0.01). Percentage breast density was also elevated (18.2%; 95% CI, 13.4 to 23.7) among the 18 women with serum mono-ethyl phthalate above the median detected level (>3.77 ng/ml) compared with women with nondetectable BPA levels (13.1%; 95% CI, 11.9 to 14.3; P_trend = 0.07). No other chemicals demonstrated associations with percentage breast density.

Conclusions

Postmenopausal women with high serum levels of BPA and mono-ethyl phthalate had elevated breast density. Further investigation of the impact of BPA and mono-ethyl phthalate on breast cancer risk by using repeated serum measurements or other markers of xenoestrogen exposure are needed.     

Genetic ablation of smooth muscle KIR2.1 is inconsequential to the function of mouse cerebral arteries

Cerebral blood flow is a finely tuned process dependent on coordinated changes in arterial tone. These changes are strongly tied to smooth muscle membrane potential and inwardly rectifying K⁺ (K_IR) channels are thought to be a key determinant. To elucidate the role of K_IR2.1 in cerebral arterial tone development, this study examined the electrical and functional properties of cells, vessels and living tissue from tamoxifen-induced smooth muscle cell (SMC)-specific K_IR2.1 knockout mice. Patch-clamp electrophysiology revealed a robust Ba²⁺-sensitive inwardly rectifying K⁺ current in cerebral arterial myocytes irrespective of K_IR2.1 knockout. Immunolabeling clarified that K_IR2.1 expression was low in SMCs while K_IR2.2 labeling was remarkably abundant at the membrane. In alignment with these observations, pressure myography revealed that the myogenic response and K⁺-induced dilation were intact in cerebral arteries post knockout. At the whole organ level, this translated to a maintenance of brain perfusion in SMC K_IR2.1^−/− mice, as assessed with arterial spin-labeling MRI. We confirmed these findings in superior epigastric arteries and implicated K_IR2.2 as more functionally relevant in SMCs. Together, these results suggest that subunits other than K_IR2.1 play a significant role in setting native current in SMCs and driving arterial tone.