Impact of maternal lifestyle factors on newborn HPRT mutant frequencies and molecular spectrum — Initial results from the Prenatal Exposures and Preeclampsia Prevention (PEPP) Study

Impact of maternal lifestyle factors on newborn HPRT mutant frequencies and molecular spectrum — Initial results from the Prenatal Exposures and Preeclampsia Prevention (PEPP) Study

Mutation Research 431 Ž1999. 279–289 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres Impact of maternal lifestyle fa...

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Mutation Research 431 Ž1999. 279–289 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres

Impact of maternal lifestyle factors on newborn HPRT mutant frequencies and molecular spectrum — Initial results from the Prenatal Exposures and Preeclampsia Prevention žPEPP/ Study William L. Bigbee a,b,c,) , Richard D. Day c,d , Stephen G. Grant a,e, Phouthone Keohavong a,c , Liqiang Xi a , Lifang Zhang a , Roberta B. Ness a

b,c

Department of EnÕironmental and Occupational Health, Graduate School of Public Health, UniÕersity of Pittsburgh, Pittsburgh, PA 15238, USA b Department of Epidemiology, Graduate School of Public Health, UniÕersity of Pittsburgh, Pittsburgh, PA 15261, USA c Cancer Epidemiology Program, UniÕersity of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA d Department of Biostatistics, Graduate School of Public Health, UniÕersity of Pittsburgh, Pittsburgh, PA 15261, USA e Magee-Women’s Research Institute, Pittsburgh, PA 15213, USA Received 19 July 1999; received in revised form 30 August 1999; accepted 30 August 1999

Abstract Epidemiological studies have demonstrated associations between maternal tobacco smoke exposure and consumption of alcohol during pregnancy and increased risk of pediatric malignancies, particularly infant leukemias. Molecular evidence also suggests that somatic mutational events occurring during fetal hematopoiesis in utero can contribute to this process. As part of an ongoing multi-endpoint biomarker study of 2000 mothers and newborns, the HPRT T-lymphocyte cloning assay was used to determine mutant frequencies ŽM f . in umbilical cord blood samples from an initial group of 60 neonates born to a sociodemographically diverse cohort of mothers characterized with respect to age, ethnicity, socioeconomic status, and cigarette smoke and alcohol exposure. Non-zero M f Ž N s 47. ranged from 0.19 to 5.62 = 10y6 , median 0.70 = 10y6 , mean " SD 0.98 " 0.95 = 10y6 . No significant difference in M f was observed between female and male newborns. Multivariable Poisson regression analysis revealed that increased HPRT M f were significantly associated with maternal consumption of alcohol at the beginning wRelative Rate ŽRR. s 1.84, 95% CI s 0.99–3.40, P s 0.052. and during pregnancy ŽRR s 2.99, 95% CI s 1.14–7.84, P s 0.026.. No independent effect of self-reported active maternal cigarette smoking, either at the beginning or throughout pregnancy, nor maternal passive exposure to cigarette smoke was observed. Although based on limited initial data, this is the first report of a positive association between maternal alcohol consumption during pregnancy and HPRT M f in human newborns. In addition, the spectrum of mutations at the HPRT locus was determined in 33 mutant clones derived from 19 newborns of mothers with no self-reported exposure to tobacco smoke and 14 newborns of mothers exposed passively or actively to cigarette smoke. In the unexposed group, alterations leading to

)

Corresponding author. Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15238, USA. Tel.: q1-412-967-6534. E-mail address: [email protected] ŽW.L. Bigbee. 0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 1 7 2 - 4

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specific exon 2–3 deletions, presumably as a result of illegitimate VŽD.J recombinase activity, were found in five of the 19 mutants Ž26.3%.; in the passively exposed group, two exon 2–3 deletions were present among the seven mutants Ž28.6%.; and in the actively exposed group, six of the seven mutants Ž85.7%. were exon 2–3 deletions. Although no overall increase in HPRT M f was observed and the number of mutant clones examined was small, these initial results point to an increase in VŽD.J recombinase-associated HPRT gene exon 2–3 deletions in cord blood T-lymphocytes in newborns of actively smoking mothers relative to unexposed mothers Ž P s 0.011.. Together, these results add to growing molecular evidence that in utero exposures to genotoxicants result in detectable transplacental mutagenic effects in human newborns. q 1999 Elsevier Science B.V. All rights reserved. Keywords: HPRT; Tobacco smoking; Alcohol; Molecular spectrum; VŽD.J recombination; Transplacental mutagenesis

1. Introduction Recent epidemiologic studies have demonstrated an association between maternal environmentalroccupational exposures, lifestyle habits, specifically cigarette smoking, alcohol consumption, and aspects of diet during pregnancy that impose excess risk of subsequent infant and pediatric malignancies in human newborns w1–9x. To investigate the potential transplacental genotoxic impact of these exposures in utero, we have undertaken a molecular epidemiology study of 2000 pregnant women and newborns applying biomarkers of exposure, genetic susceptibility, and early biological effects measured in maternal and umbilical cord blood specimens ŽTable 1.. This comprehensive investigation follows our initial survey of somatic mutation in newborns in which we found that maternal exposure to tobacco smoke andror maternal lifestyle factors associated with lower socioeconomic status appeared to increase the frequency and alter the spectrum of the molecular mechanisms of somatic mutation in utero w10x. Previous studies of the effects of maternal lifestyle factors and exposures during pregnancy on human newborn genotoxicity have produced equivocal evidence for the impact of tobacco smoke exposure on HPRT M f w10–13x; the most recent and provocative study w14x suggests an association between passive maternal exposure to tobacco smoke and an increase in specific HPRT locus exon 2–3 deletion mutations in umbilical cord blood T lymphocytes mediated by VŽD.J recombinase, the enzyme system responsible for generation of functional immunoglobulin and T-cell receptor diversity through rearrangement of multiple variable ŽV., diversity ŽD. and junctional ŽJ. genomic sequences. As an initial report from our ongoing study exploring these effects in human newborns, here we provide results from the first 60

neonates in the study that indicate that maternal alcohol consumption during pregnancy is positively associated with increased newborn HPRT M f measured in umbilical cord blood T lymphocytes and, consistent with the recent earlier report, that maternal exposure to tobacco smoke results in an increase in

Table 1 Prenatal Exposures and Preeclampsia Prevention ŽPEPP. study biomarkers Exposure biomarkers Ø Serum cotinine Ø 4-aminobiphenyl-hemoglobin Ž4-ABP-Hb. adducts Genetic susceptibility biomarkers — phase Ir II xenobiotic metabolizing enzyme polymorphisms a Ø Genotyping panel CYP1A1 CYP2E1 GSTM1 GSTT1 NAT2U Ø mRNA phenotyping panel CYP1A1 CYP2E1 CYP1B1 Effect biomarkers Ø gpa locus Vf in erythrocytes Ø HPRT locus M f and mutational spectrum in T-lymphocytes Ø HPRT locus VŽD.J recombinase-mediated deletions in T-lymphocytesb Ø Single cell gel electrophoresis ŽSCGE. ŽComet. assay in peripheral blood mononuclear cells Ø FISH-based chromosome translocations and bleomycin sensitivity in T-lymphocytesc a

In collaboration with Dr. M. Romkes, University of Pittsburgh. b In collaboration with Drs. J. Fuscoe and J. Scheerer, EPA National Health and Environmental Effects Research Laboratory. c In collaboration with Dr. J. Tucker, Lawrence Livermore National Laboratory.

W.L. Bigbee et al.r Mutation Research 431 (1999) 279–289

the class of characteristic deletion mutations associated with illegitimate VŽD.J recombinase activity.

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consents and blood samples were obtained as approved by the University of PittsburghrMWH IRBs. 2.3. HPRT T-lymphocyte cloning assay

2. Materials and methods 2.1. Prenatal Exposures and Preeclampsia PreÕention (PEPP) Study design The data presented here are initial results from an ongoing prospective molecular epidemiologic study in which approximately 2000 pregnant women presenting for care at Magee Women’s Hospital ŽMWH. of Pittsburgh Prenatal Clinics and MWH-associated private OBrGYN practices are being recruited and enrolled. This study group was selected to accurately sample the broad spectrum of sociodemographic diversity represented in the MWH patient population. Study subjects are being recruited from healthy women aged 14–44 who have a pregnancy of typically 10–14 weeks gestation. Subjects are consented and enrolled, using protocols approved by the University of PittsburghrMWH Institutional Review Boards ŽIRBs., at their initial prenatal visit at which time a comprehensive interview questionnaire is administered to capture and quantitate, together with abstracted data from clinical records, maternal factors regarding socioeconomic status, exposure to tobacco smoke Žactive and passive. and consumption of alcohol Žbeer, wine, or hard liquor., together with other demographic and lifestyle variables and exposures. Maternal peripheral blood samples are being obtained at enrollment, at 22–28 weeks gestation, at predelivery, and 1–2 days postpartum. A sample of umbilical cord blood from each newborn is also collected. Lastly, a postpartum interview questionnaire is administered to capture maternal variables during the last trimester of the pregnancy. 2.2. Subjects and samples Eligible maternal subjects were pregnant women between 14 and 44 years of age, in generally good health and at 22 weeks of gestation or less at enrollment as dated by the last menstrual period. Umbilical cord blood samples were obtained from healthy newborns. Maternal peripheral blood and umbilical cord blood samples were drawn into sodium heparin anticoagulant, stored at room temperature and delivered to the laboratory within 48 h of collection. Patient

The T-cell cloning assay system used to determine HPRT M f was a modification of previously published protocols w15,16x. Mononuclear cells were isolated from 10–20 ml of heparinized umbilical cord blood with Ficoll-Hypaque Plus ŽPharmacia Biotech, Piscataway, NJ.. To reduce contamination by red blood cells, 500 ml of Red-Out ŽRobbins Scientific, Sunnyvale, CA., a murine monoclonal antibody specific to a human erythroid cell lineage carbohydrate epitope, was added per 10 ml of blood during the isolation. The washed mononuclear cells were cryopreserved in freezing medium containing 10% fetal bovine serum ŽFBS., 8% dimethyl sulfoxide ŽDMSO, Sigma, St. Louis, MO., and 82% RPMI 1640 using a NALGENEe Cryo 18C freezing container placed in a y808C freezer to achieve an approximate 18Crmin rate of cooling, then stored at y1508C. Rapidly thawed mononuclear cells from the cryopreserved samples were washed once with RPMI 1640 and incubated in medium containing 10% FBS, 20% HL-1, 1% penicillin-streptomycin, 1% glutamine and 1 mgrml phytohemagglutinin ŽPHA-P, Difco Laboratories, Detroit, MI. at 378C in 5% CO 2 for 36–40 h. The cells were then washed, counted, and plated in 96-well round-bottomed plates at cell densities of one and five cellsrwell in non-selection medium and 2 = 10 4 cellsrwell in 10 mM 6-thioguanine Ž6-TG. selection medium. The cells were plated in 200 ml of RPMI 1640 medium with 10% FBS, 10% human T-STIMe with PHA ŽCollaborative Biomedical Products, Bedford, MA., 20% HL-1, 1% penicillin-streptomycin, 1% glutamine and HPRT y 36 = 4 cells Žkindly supplied by H. Liber. at 2 = 10 4 cellsrwell as feeder cells. The 36 = 4 feeder cells were irradiated at 10 7 cells in one ml RPMI 1640 with no serum to a dose of 18 Gy with a 137 Cs source. Each sample was plated using two plates at one cellrwell and two plates at five cellsrwell in non-selection medium, and six plates in selection medium. The plates were incubated at 378C in a humidified 5% CO 2 incubator for 14–21 days. Growing T-cell clones were scored using an inverted microscope.

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W.L. Bigbee et al.r Mutation Research 431 (1999) 279–289

Cloning efficiencies ŽCE. with or without 6-TG were calculated using the Poisson relationship, Po s eyx where Po is the observed fraction of the wells with no detectable cell growth and x is the average number of cells inoculated per well; the CE is then calculated using the equation CE s yln Porx. The mutant frequencies ŽM f . were calculated as the ratio of the mean CE in the presence Žselection plates. and absence Žnon-selection plates. of 6-TG. Individual mutant clones were expanded in 2 ml of selective media with feeder cells for another seven days for HPRT molecular spectrum analysis. 2.4. HPRT molecular analyses Each TG r clone isolated in the T-cell cloning assay as described above was expanded in selective medium, distributed into samples of 10 4 cells in 0.5 ml Eppendorf tubes, and stored at y808C until molecular analysis. One randomly selected clone from each of 33 of the total of 47 newborns with non-zero M f was successfully expanded and analyzed. 2.4.1. RT-PCR and cDNA analysis Cells from each selected clone were taken from the y808C freezer and thawed, the cells were spun down and the cell pellet was first washed gently with 50 ml PBS, then resuspended and lysed in 5 ml of cDNA synthesis mix Ž50 mM Tris–HCl pH 8.3; 75 mM KCl; 3 mM MgCl 2 ; 10 mM DTT; 500 mM dNTP; 0.1 ngrml BSA; 1 unitrml RNasin; 10 ngrml IGEPAL; 2.5 unitsrml M-MmLART. with incubation at 378C for 1 h. A 2.5 ml aliquot of the mix was then diluted in a final 25 ml PCR reaction mix containing 10 mM Tris–HCl, pH 8.3; 50 mM KCl, 15 mM MgCl 2 , 0.01% gelatin, 300 mM dNTP; 0.2 mM each of primers p1 Ž5X-CTGCTCCGCCACCGGCTTCC-3X ; nucleotides 1617 1636 of the human HPRT gene. and p2 Ž5X-GATAATTTTACTGGCGATGT-3X ; nucleotides 41 565 41 546 of the human HPRT gene., and 2 units of Taq DNA polymerase, heated to 948C for 4 min, followed by 25 cycles of PCR Ž948C 1 min, 558C 45 s, 728C 2 min.. One microliter of the above PCR reaction mixture was then used for a second round of PCR using the same PCR reaction mixture as above except primers p1 and p2 were replaced by primers p3 Ž5XCCTGAGCAGTCAGCCCGCGC-3X ; nucleotides

™ §

™ §

1641 1660 of the human HPRT gene. and p4 Ž5X-CAATAGGACTCCAGATGTTT-3X ; nucleotides 41 545 41 526 of the human HPRT gene., for 33 PCR cycles. The PCR products were analyzed on a 5% PAGE gel; the appropriate bands were cut out, the cDNA extracted and then sequenced by ABI automated DNA sequencing analysis. 2.4.2. Multiplex PCR amplification and analysis Clones that indicated the presence of large deletion by cDNA analysis were further characterized by multiexon PCR to identify the deleted genomic sequence. Aliquots from the cell samples were used as the source of crude genomic DNA. Briefly, 1 = 10 4 cells were washed with PBS buffer and resuspended in 50 ml of lysis buffer containing 6.7 mM MgCl 2 , 16.6 mM ŽNH 4 . 2 SO4 , 6.8 mM EDTA, 67 mM Tris– HCl, pH 8.8, 5 mM 2-mercaptoethanol, 0.45% IGEPAL, 0.45% Tween 20, and 100 mgrml proteinase K, and incubated for at least 1 h at 568C. The proteinase K was then inactivated by heating at 968C for 10 min. Five microliters of this crude DNA was mixed with 20 ml of a PCR mixture containing seven pairs of primers to amplify each of the HPRT exons 2–9 w17x, 6.7 mM MgCl 2 , 16.6 mM ŽNH 4 . 2 SO4 , 6.8 mM EDTA, 67 mM Tris–HCl, heated to 948C for 4 min, followed by 35 PCR cycles Ž688C for 2 min, 948C for 1 min and 598C for 1 min.. Exon 1 was amplified separately by primers pE1 Ž5X-AGCTTCAGGCGGCTGCGACGAGCCCTCAGG-3X nucleotides 1530 1559 of the human HPRT gene. and pE2 Ž5X-CGGCCGCCCGAGCCCGCACTGCGGATCCCG-3X nucleotides 1804 1775 of the human HPRT gene.. Five microliters of the PCR products were then analyzed on a 5% PAGE gel.



§

2.5. Statistical methods Descriptive statistics and simple univariate procedures exploring the underlying distributions of the data and associations of HPRT M f with selected maternal variables were carried out using MINITABe software. Exact statistical tests, using STATXACTe software, were carried out for binomial comparisons of categorical data where sample sizes made the use of the x 2 test inappropriate. Multivariable Poisson regression was performed using EGRETe statistical software. The contribution of individual maternal factors to the final regression

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models were assessed using the likelihood ratio statistics with a critical value of P s 0.05. The significance of different levels of HPRT M f within individual factors was assessed using a z-statistic, the Wald test statistic. Coefficients, standard errors and rate ratios ŽRR. of HPRT M f were calculated for the final regression models. P values were not adjusted for multiple comparisons and the models presented have included only the main subset of maternal factors wage, ethnicity, education and family income as measures of SES, tobacco smoke exposure Žactive and passive., and alcohol consumption Žbeer, wine, and hard liquor combined as estimated total ounces.x to be evaluated in the study. It is therefore important to note that these inferences are drawn only from the initial experimental results of the study and an exploratory data analysis restricted to the selected set of maternal sociodemographic variables. 3. Results 3.1. Maternal sociodemographic characteristics An initial cohort of 60 healthy newborns Ž27 female and 33 male neonates. from the PEPP study population was selected for determinations of HPRT M f and statistical analysis for associations with maternal sociodemographic variables. A summary of the maternal factors considered in the analysis is

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presented in Table 2. The specific maternal variables included maternal age, ethnicity, family income and educational level as measures of SES, tobacco smoke exposure Žactive and passive., and alcohol consumption prior to and during pregnancy together with newborn gender. The population is approximately one-third African-American and two-thirds Caucasian-American and consists primarily of young women Žmedian age, 20 years; mean age " SD, 22.5 " 4.7 years.. These women were enrolled in the study at a mean gestational age of 10.1 " 3.8 weeks. As detailed in Table 2, the African-American and Caucasian-American mothers differed substantially in regard to the distribution of SES measures, and their self-reported prevalence and level of active cigarette smoking both at the time of the initial baseline interview and during the pregnancy ŽTable 3.. The overall prevalence of active cigarette smoking among these women at the time of enrollment was 45.0%. Among the smoking subjects, 48.2% self-reported that they continued to smoke only early in pregnancy and 51.8% reported continued smoking through the last trimester. Smoking prevalence was similar between the Caucasian-American and the African-American women Ž40.5% and 50.0%, respectively.; however, the Caucasian women reported smoking more cigarettes per day than the AfricanAmerican women both early and late in pregnancy

Table 2 Summary of PEPP maternal subjects sociodemographic data Maternal factor

African-American

Caucasian-American

Other a

Overall

Age Žmean " SD. Žrange. Ethnicity Family income - $10 K $10–20 K $20–35 K ) $35 K Don’t know Education Less than grade 12 Diplomaq GED Some college BS or greater

20.6 " 3.7 14–30 22 Ž36.7%.

23.7 " 4.9 17–34 37 Ž61.7%.

17 – 1 Ž1.7%.

22.5 " 4.7 14–34 60

7 Ž31.8%. 6 Ž27.3%. 1 Ž4.6%. 1 Ž4.6%. 7 Ž31.8%.

10 Ž27.0%. 9 Ž24.3%. 8 Ž21.6%. 9 Ž24.3%. 1 Ž2.7%.

1 Ž100%. – – – –

18 Ž30.0%. 15 Ž25.0%. 9 Ž15.0%. 10 Ž16.7%. 8 Ž13.3%.

5 Ž22.7%. 14 Ž63.6%. 3 Ž13.6%. 0 Ž0%.

3 Ž8.1%. 15 Ž40.5%. 11 Ž29.7%. 8 Ž21.6%.

1 Ž100%. – – –

9 Ž15.0%. 29 Ž48.3%. 14 Ž23.3%. 8 Ž13.3%.

a

AsianrPacific Islander.

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Table 3 Summary of newborn HPRT M f by maternal sociodemographic factors Factor Overall Maternal age Žyears. - 20 20–30 ) 30 Ethnicity African-American Caucasian-American Other Maternal education Less than grade 12 Diplomaq GED Some college BS or greater Family income Ž$K. - $10 K $10–20 K $20–35 K $35 K Unknown Newborn gender Female Male Active cigarette smoking Never Quit before pregnancy Quit at pregnancy Continued during pregnancy Alcohol consumptiond None Quit at pregnancy Continued during pregnancy

N Ž n. a

Mean CE

Median M f Ž=10y6 . .c

Mean M f " SD b Ž=10y6 . 0.98 " 0.95

60 Ž13.

0.37

0.70 Ž0.52

19 Ž3. 35 Ž8. 6 Ž2.

0.32 0.41 0.26

0.76 Ž0.64. 0.67 Ž0.45. 0.57 Ž0.27.

0.84 " 0.40 1.10 " 1.20 0.69 " 0.54

22 Ž3. 37 Ž10. 1 Ž0.

0.32 0.40 0.21

0.60 Ž0.56. 0.76 Ž0.45. 1.09

1.04 " 1.31 0.93 " 0.63 1.09

9 Ž1. 29 Ž6. 14 Ž4. 8 Ž2.

0.30 0.42 0.27 0.41

0.97 Ž0.85. 0.54 Ž0.45. 0.79 Ž0.54. 1.07 Ž0.52.

1.02 " 0.41 0.74 " 0.69 1.41 " 1.61 1.12 " 0.80

18 Ž4. 15 Ž3. 18 Ž4. 1 Ž0. 8 Ž2.

0.32 0.47 0.35 0.17 0.31

0.66 Ž0.52. 0.55 Ž0.45. 0.66 Ž0.34. 5.62 0.84 Ž0.76.

0.77 " 0.41 0.92 " 0.55 0.80 " 0.59 5.62 1.22 " 1.08

27 Ž9. 33 Ž4.

0.34 0.38

0.79 Ž0.41. 0.67 Ž0.60.

1.06 " 1.22 0.93 " 0.75

21 Ž4. 12 Ž2. 13 Ž6. 14 Ž1.

0.40 0.34 0.39 0.32

0.60 Ž0.48. 0.86 Ž0.80. 0.79 Ž0.26. 0.64 Ž0.61.

0.86 " 0.80 1.40 " 1.56 1.01 " 0.82 0.78 " 0.48

39 Ž8. 18 Ž4. 3 Ž1.

0.39 0.34 0.28

0.60 Ž0.45. 0.84 Ž0.75. 2.54 Ž1.66.

0.71 " 0.48 1.34 " 1.36 2.54 " 1.24

a

Number of samples below the detectable level Žtreated as M f s 0. in each category. Mean M f " SD with M f s 0 values excluded. c Median M f with M f s 0 values included. d Significant in multivariable Poisson regression model.

b

Žmean " SD, 16.0 " 9.1 versus 11.0 " 9.0, and 9.5 " 6.2 versus 5.8 " 7.0, respectively.. As summarized in Table 3, 65.0% of the women reported never to have consumed alcoholic beverages; 30.0% reported alcohol consumption prior to and early in pregnancy but quit when they learned they were pregnant; and 5.0% who continued drinking during the pregnancy. Since subjects who consumed alcohol might also be more likely to have smoked cigarettes thus potentially confounding the effects of alcohol, the prevalence of smoking among the subjects within these categories was examined in

detail. There did not appear to be strong evidence for an increased prevalence of active smoking in the alcohol consuming group; among women who reported no alcohol use, eight of 39 Ž20.5%. were active smokers; those who quit early in pregnancy or continued drinking, four of 18 Ž22.2%. and one of three Ž33.3%., respectively, were active smokers. 3.2. Summary descriptions of newborn HPRT mutant frequencies (M f ) A total 60 newborn umbilical cord blood samples were analyzed for HPRT locus M f . These neonates

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were born to a sociodemographically diverse cohort of mothers characterized with respect to age, ethnicity, SES, and cigarette smoke and alcohol exposure. The observed newborn CE and M f stratified by these maternal factors and newborn gender are summarized in Table 3. Overall, non-zero M f Ž N s 47. ranged from 0.19 to 5.62 = 10y6 with a median and mean " SD M f of 0.70 = 10y6 and 0.98 " 0.95 = 10y6 , respectively Thirteen samples yielded observed M f of zero; when these zero values were included in the distribution, the mean M f was 0.52 = 10y6 . No significant difference in M f was observed between female and male newborns. These descriptive univariate statistics revealed no significant differences in newborn M f among women of varying SES measured by maternal education or family income nor among women who smoked cigarettes before or during pregnancy. Significant associations were observed between newborn M f and maternal age and alcohol consumption during pregnancy ŽTable 3., but since these maternal factors may have been confounded with ethnicity, SES, and smoking, these results were explored in a full multivariable Poisson model. 3.3. Maternal lifestyle factors affecting newborn HPRT mutant frequencies (M f ) With all of the selected maternal factors included in a multivariable Poisson regression analysis ŽTable 4., the strongest association of newborn HPRT M f was with maternal alcohol consumption during pregnancy. Although the number of women in this group is small Ž N s 17., the results are statistically significant both for maternal consumption of alcohol at the Table 4 Multivariable Poisson regression modela for the effect of maternal alcohol consumption during pregnancy on newborn HPRT M f Factor Alcohol consumption None Quit at pregnancy Continued during pregnancy

Coefficient

P

RR

95% CI

0.6097 1.095

0.052 0.026

1.00 b 1.84 2.99

0.99–3.40 1.14–7.84

a Model includes maternal age, ethnicity, SES, and tobacco smoking Žactive and passive.. b Referent group.

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beginning ŽRR s 1.84, P s 0.05. and during pregnancy ŽRR s 2.99, P s 0.03.. The model also revealed no independent effect of active maternal cigarette smoking, either at the beginning or throughout pregnancy, nor maternal passive exposure to cigarette smoke. These results in the model appear to be stable as maternal age, ethnicity, SES measured by maternal education or household income did not significantly impact the observed M f nor significantly confound the observed alcohol and tobacco smoke exposure observations. Among several previous studies that have investigated the potential impact of maternal lifestyle factors and exposures on newborn HPRT M f , this is the first report of a significant and positive association of maternal alcohol use during pregnancy with increased HPRT M f in human newborns. 3.4. Association of maternal exposure to tobacco smoke during pregnancy and newborn HPRT molecular spectrum A total of 33 6-TG resistant putatively mutant clones, one each from 19 newborns of mothers with no self-reported exposure to tobacco smoke and 14 from newborns of mothers exposed actively or passively to cigarette smoke were molecularly characterized to describe the HPRT mutational spectrum in these neonates. The initial analysis focused on the association between in utero exposure to tobacco smoke and the subset of fetal mutations resulting from illegitimate genomic deletions mediated by VŽD.J recombinase, since a previous study had suggested an increase in these specific mutational events in newborns of mothers passively exposed to tobacco smoke during pregnancy w14x. Multiexon PCR amplification of the HPRT gene DNA, and RT-PCR amplification and cDNA sequencing of the HPRT gene mRNA from these clones revealed that a substantial fraction overall Ž13 of 33, 39.4%. resulted from HPRT locus exon 2–3 deletions characteristic of mutations mediated by illegitimate VŽD.J recombination. When these results were stratified with respect to maternal tobacco smoke exposure during pregnancy, in the unexposed group, exon 2–3 deletions accounted for five of 19 mutants Ž26.3%.. In the combined exposed group Žpassive plus active.,

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eight exon 2–3 deletions Ž57.1%. were present among the 14 mutants. This effect appeared to be driven predominantly by an increase in the actively exposed group where six of the seven mutants Ž85.7%. were exon 2–3 deletions. These initial results point to a significant increase in putative VŽD.J recombinase-associated HPRT gene exon 2–3 deletions in cord blood T-lymphocytes in newborns of actively smoking mothers relative to unexposed mothers Ž P s 0.011., suggesting that these events may be induced by in utero exposures to tobacco smoke-derived genotoxicants.

4. Discussion The results presented here represent initial data from the University of PittsburghrMagee Women’s Hospital PEPP Study, an ongoing molecular epidemiologic investigation designed to assess the potential genotoxic impact to the developing fetus of maternal exposures including tobacco and alcohol use, diet, vitamin use, and X-ray exposure during pregnancy. The continuing PEPP study will ultimately enroll and study 2000 newborn and maternal subjects and will also determine cotinine levels and 4-aminobiphenyl hemoglobin Ž4-APB-Hb. adducts in maternal and newborn samples as independent exposure biomarkers to more accurately and quantitatively classify newborn cigarette smoke exposure. Phase IrII maternal and newborn metabolic enzyme polymorphisms conferring potential genetic susceptibility to cigarette smoke and alcohol are also being determined and will be analyzed to investigate potential gene-environment interactions in modulating the effect biomarkers employed in the study — gpa Vf , HPRT M f , and chromosome translocations — measured in umbilical cord blood cells. As reported here, the initial results from this study suggest, for the first time, that maternal alcohol consumption during pregnancy appears to be associated with an increase in newborn HPRT M f . This association does not appear to be confounded by tobacco smoke exposure in the alcohol consuming mothers since these subjects are no more likely to be active smokers than non-drinking mothers and the effect of alcohol consumption is not reduced in the multivariable Poisson regression when smoking Žac-

tive or passive. is included in the model. This result is of particular interest in the context of a recently published epidemiologic study that reported maternal alcohol consumption during pregnancy was associated with an increased risk of infant leukemia ŽOR s 1.43 for ALL, 2.64 for AML, and 1.60 overall. w9x. Maternal consumption of alcoholic beverages may result in fetal exposure to acetaldehyde, a genotoxic metabolite formed by in vivo ethanol oxidation. Acetaldehyde has been demonstrated to induced HPRT locus mutations in human T-lymphocytes in vitro w18x and has been associated with the formation of intrastrand crosslinks resulting in characteristic tandem GG to TT base substitutions in a shuttle vector mutation system propagated in SV-40 transformed WI38-VA13 human fibroblasts w19x. Alternatively, alcohol associated genotoxicity may proceed through indirect mechanisms. In animal systems, ethanol has been shown to induce microsomal cytochrome P450 enzymes, potentially leading to an increased production in vivo of electrophilic genotoxic metabolites of exogenous andror endogenous promutagens in placental andror fetal tissues w20–22x. Taken together, these results add molecular evidence that in utero exposure to alcohol or its metabolites may result in quantifiable mutagenicrleukemogenic effects in human newborns. No significant independent effect of maternal exposure to tobacco smoke, either active or passive, during pregnancy on newborn HPRT M f was observed in this group, a result that is concordant with three previously published studies that did not detect an increase of HPRT M f in newborns of mothers who smoked cigarettes during pregnancy w10,11,13x. A single study reported an elevation in HPRT M f of neonates born to smoking mothers w12x. Although no overall effect of maternal exposure to tobacco smoke on newborn HPRT M f was apparent, there appeared to be a significant increase in the prevalence of newborn HPRT gene deletion mutations associated with illegitimate VŽD.J recombinase activity with active maternal tobacco smoke exposure during pregnancy. VŽD.J mediated recombination is the genetic mechanism responsible for the generation of functional T-cell receptor genes and immunoglobulin genes in lymphoid cells. However, illegitimate VŽD.J recombination occurring at other genetic loci has been shown to result in a number of chromosome

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translocations which activate oncogenes and, in some cases, inactivate tumor suppressor genes w23,24x, which appear to be associated with specific pediatric lymphoid malignancies w25–30x. Since this same mechanism also generates a characteristic exon 2–3 deletion resulting in inactivation of the HPRT gene w31x, this subclass of HPRT mutants may serve as a biomarker of illegitimate VŽD.J mediated recombination in peripheral blood T-lymphocytes. These deletion mutants comprise a substantial fraction Ž; 40%. of the total HPRT gene mutations observed in the T-lymphocytes of human newborns w31,32x. As pointed out by Finette et al. w32x, the age-specific peak of VŽD.J recombinase mediated HPRT locus deletions in newborns and young children correlates with the observed age-incidence distribution of acute lymphocytic leukemia in which illegitimate VŽD.J recombination plays a critical mechanistic role. Consistent with the earlier report of Finette et al. w14x, the molecular spectrum of HPRT gene mutations observed in this initial group of PEPP study newborns suggests an association between the relative prevalence of the subclass of VŽD.J recombinase mediated exon 2–3 HPRT gene deletions and maternal tobacco smoke exposure during pregnancy. The presently reported data extends the initial finding of Finette et al. w14x in regard to the impact of maternal passive exposure to actively smoking mothers. It is important to emphasize that the conclusions reached in this initial report regarding associations between maternal tobacco smoke exposures and HPRT M f and the molecular spectrum of HPRT locus mutations are based on maternal self-reports. Since self-reporting has been shown to underestimate true tobacco smoke exposures, particularly among pregnant women, the ongoing PEPP study is employing two biomarkers of tobacco smoke exposure, serum cotinine and 4-ABP-Hb adducts, to more accurately classify and quantitate these exposures. The addition of these exposure biomarker data will be invaluable for the accurate classification of these subjects and for the refinement of these initial observations. Lastly, the PEPP Study includes the characterization of maternal and newborn phase IrII metabolic enzyme genotypes and expression phenotypes. The level of genotoxic damage in newborn cells is likely to be modulated by the expression of procarcinogen

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metabolizing CYP and phase II enzymes which contribute to the metabolic activation or detoxification of chemical agents, for example, a number of tobacco-derived chemicals including polycyclic aromatic hydrocarbons and nitrosamines. The majority of these carcinogenic compounds are not only metabolized by the CYPs but also induce CYP enzyme expression and activity. In addition, genetic polymorphisms resulting in altered activity levels have also been identified for several human CYP and phase II enzymes. Therefore, metabolizing enzyme genotype and the levels of CYP expression may help to explain interindividual susceptibility to genotoxic damage. Indeed, results from this initial group of PEPP study subjects indicate that the presence or absence of ‘‘at-risk’’ combinatorial genotypes is significantly associated with elevated or decreased gpa Vf and HPRT M f in maternal and newborn cells w33x. The ongoing PEPP study will provide a substantial database for confirmation and extension of the initial results presented here and also permit the investigation of important gene-environment interactions revealed by both the level and molecular spectrum of somatic mutations observed in human newborns. 5. Conclusion The frequency and molecular spectrum of HPRT locus mutations in umbilical cord blood T-lymphocytes was determined in an initial group of 60 healthy newborns of women recruited in the University of PittsburghrMagee-Women’s Hospital PEPP Study. Analysis of the impact of maternal sociodemographic factors and exposures on newborn HPRT M f suggested, for the first time, a significant positive association with maternal consumption of alcohol during pregnancy. In addition, an increased prevalence of VŽD.J recombinase mediated HPRT exon 2–3 deletion mutations was observed in newborns of mothers actively exposed to tobacco smoke during pregnancy. Acknowledgements This research was supported by grant number 1 R01 HD33016 from the National Institute of Child

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Health and Human Development to WLB. We acknowledge the valuable contributions of the PEPP Core Group of clinical collaborators and the Labor and Delivery staff of Magee Women’s Hospital of Pittsburgh for recruitment of subjects and the collection of maternal and umbilical cord blood samples.

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