1. Gender-specific protective effect of hemoglobin on arsenic- induced skin lesions David C. Christiani,MD,MPH,MS Dhaka, Bangladesh January 4, 2006

2. Harvard-DCH Collaborators Carrie V. Breton, Andres Houseman, Molly Kile, Quazi Quamruzzaman, Mahmuder Rahman, Golam Mahiuddin, David C. Christiani. Dhaka Community Hospital, Dhaka, Bangladesh and Harvard School of Public Health, Boston, Mass., USA

3. Background Chronic arsenic poisoning is a major health concern in Bangladesh and in other regions of the world. Mechanisms of human toxicity remain elusive, especially dermatologic outcomes. In vitro and in vivo studies show that inorganic arsenic binds to hemoglobin and can change cell morphology, heme metabolism, and hemoglobin levels.

4. Background Acute exposure to arsernite has been shown to cause anemia, leukopenia, and throbocytopenia from bone marrow suppression. Arsine gas can cause hemolytic anemia. Arsenic-induced skin lesions, cancerous lesions, have less hemoglobin than do non- cancerous lesions.

5. Background Chronic arsenic ingestion alters heme metabolism and increases excretion of total porphyrins. However, the effect of chronic As exposure on the hemologic system remains relatively unexplored, with no direct evidence of As related anemia.

6. Hemoglobin Hemoglobin (Hgb) is of particular interest because of its use in the diagnosis of anemia. Anemia is defined as a blood Hgb below 12g/dL. Bangladesh: National prevalence of anemia has remained constant at 74% for the past 30 years. Iron deficiency due to inadequate intake and low dietary bioavailability of iron.

7. Aims To assess the relationship between blood Hgb and skin lesions in a case-control study of 1,800 persons in Pabna. To assess potential interactions between AS, Hgb, and genetic polymorphisms in genes important for detoxification. To examine direct associations between toenail and urinary arsenic levels and As in a disease-free, repeated measures study of 184 adults from 50 families is Pabna.

8. Methods Case-Control Study:2001-2003, 900 case-control pairs (n=1,800) recruited Physician-diagnosed lesions; keratosis, spotted melanosis, Bowen’s disease, SCC Separate study: 248 persons (148 adults) from 50 families (25 in suspected high, 25 in suspected low exposure regions) for a four time per year sampling over 2001-2005. “Dosimetry Study”

9. Data collection and analysis Hgb determined by Sahli’s method. Toenail clippings- Total Inorganic As analyzed by ICP-MS. Well-water sampling after several minute flush. Blood for DNA – PCR for GSTM1, GSTT1, GSTP1.

10. Statistical Analysis Descriptive statistics (Chi square, t-test,Wilcoxon rank sum), comparing Cases vs Controls; and characteristics of dosimetry group Conditional logistic regression to examine Hgb-skin lesions while adjusting for co-variation (age, gender, smoking, betel-nut chewing, etc.). For repeated measures, separate regressions (mixed models) with a random family effect.

11. Table1. Sociodemographic characteristics of 1800 cases and controls CasesControlsP-value Physical Male61.7%61.8%0.96 * Mean age (years)33.533.30.76 + Mean body mass index20.020.20.05 + Social Education<0.0001 * no school (illiterate)20.5%15.0% no school (able to write)30.4%24.7% primary12.2%13.0% Secondary32.6%41.4% college +4.3%5.9% Chew tobacco14.8% 0.96 * Chew betel nuts28.6%23.9%0.03 * Ever smoked28.6%31.6%0.17 *

12. Table1 (cont.) Sociodemographic characteristics of 1800 cases and controls CasesControlsP-value Demographic Hemoglobin categories0.0175 * Hb <12 g/dl (anemic)13.7%10.5% Hb between12.1 - 14.071.0%70.1% Hb between 14.1 - 18.015.4%19.4% Median water arsenic(ug/l)3911.4<0.0001 § Median toenail arsenic (ug/g)3.71.6<0.0001 § * Chi square test + Student t- test § Wilcoxon Rank Sum test

13. Table 2. Results from a penalized conditional logistic regression spline model with the outcome skin lesions. The estimates for Hb are valid above a level of 12 g/dL (n=1708 ). 95% CI ORlowerupper Hb (threshold at 12 g/dl)0.790.690.91 BMI1.010.971.05 Education no school (able to write)1.180.801.73 primary0.810.501.30 Secondary0.480.300.74 college +0.900.441.85 Smoked cigarettes0.800.531.22 Smoked bidi0.480.310.74 Chewed betelnuts1.441.002.09 Chewed tobacco0.490.310.78 Logged toenail arsenic1.791.522.10

14. Table 3 (cont.) Results from a penalized conditional logistic regression spline model with the outcome skin lesions and a gender by Hb interaction (n=1708). The estimates for Hb are valid above a level of 12 g/dL. 95% CI ORlowerupper Hemoglobin (threshold at 12)1.160.921.46 BMI1.010.961.05 Education no school (able to write)1.240.831.85 no school (primary)0.840.521.38 Secondary0.450.280.72 college +0.350.170.73

15. Smoked cigarettes0.770.491.19 Smoked biri0.450.280.71 Chewed betelnuts1.501.022.19 Chewed tobacco0.480.300.77 Logged toenail arsenic1.821.542.15 Gender-Hb interaction0.520.380.70 Spline (logged water arsenic) Linear1.131.041.23 non-linear (p value <.0001) Table 3 (cont) Results from a penalized conditional logistic regression spline model with the outcome skin lesions and a gender by Hb interaction (n=1708). The estimates for Hb are valid above a level of 12 g/dL. 95% CI ORlowerupper

16. Table 4. Summary statistics describing the physical and sociodemographic characteristics of the repeated measures population (N=184). Physical Gender (%) Male 45.1% Female 54.9% Age (years) 35.7 Body mass index 20.8

17. Demographic Mean hemoglobin (g/dL) 13.7 Median averaged As toenail (μg/g) 0.879 Median total urinary As (ppb) 29.8 Table 4. Summary statistics describing the physical and sociodemographic characteristics of the repeated measures (“dosimetry”) population (N=184). Social Smoking24.5% Education no school (illiterate)16.9% no school (able to write)31.5% primary20.1% Secondary25.0% college +6.5%

18. Table 5. Results of a mixed linear regression model with the outcome Hb (g/dL). β coefSEP value Logged average toenail arsenic-0.1070.1460.465 Age-0.0060.0080.490 BMI0.0690.0330.040 Gender (Male)2.2340.267<.0001 Education no school (able to write)0.0740.3230.820 primary-0.2310.3670.531 Secondary0.0010.3670.998 college +-1.0490.5240.048 Smoked cigarettes-0.1570.3120.620

19. Conclusions Low hemoglobin may exacerbate the detrimental health effects of chronic arsenic poisoning. While providing clean water remains the primary solution to the arsenic epidemic, improving nutrition and reducing iron deficiency may ameliorate the adverse health effects of arsenic.

20. Acknowledgments We thank our colleagues, technicians, laboratory, and administrative staff at Dhaka Community Hospital and the Pabna Community Clinic in Bangladesh. We also acknowledge the academic assistance of Tom Smith and Paul Catalano, and the technical expertise of Janna Frelich, Thomas Can Geel, Ian James, Li Su, Ema Rodrigues and Meredith Jones.

21. Grant Support This publication was made possible by NIH grants T32 ES07069, research grants ES011622, ES05947, and center grant # ES00002. Its contents are solely the responsibility of the authors and do not represent the official views of the NIH.