Research Creatinine, Diet, Micronutrients, and Arsenic Methylation in West Bengal, India Arin Basu,1,2 Soma Mitra,3 Joyce Chung,1,4 D.N. Guha Mazumder,5 Nilima Ghosh,5 David Kalman,6 Ondine S. von Ehrenstein,1,7 Craig Steinmaus,1,8 Jane Liaw,1 and Allan H. Smith1 1Arsenic Health Effects Research Group, School of Public Health, University of California, Berkeley, California, USA; 2Health Sciences Centre, University of Canterbury, Christchurch, New Zealand; 3School of Human Sciences, London Metropolitan University, London, United Kingdom; 4Veteran’s Administration, Palo Alto Health Care System, Palo Alto, California, USA; 5Department of Gastroenterology, Institute of Post Graduate Medical Education and Research, Kolkata, India; 6Department of Environmental Health, University of Washington, Seattle, Washington, USA; 7School of Public Health, University of California, Los Angeles, California, USA; 8Office of Environmental Health Hazard Assessment, California Environmental Protection Agency, Oakland, California, USA Background : Ingested inorganic arsenic (InAs) is methylated to monomethylated (MMA) and dimethylated metabolites (DMA). Methylation may have an important role in arsenic toxicity, because the monomethylated trivalent metabolite [MMA(III)] is highly toxic. o Bjectivs : We assessed the relationship of creatinine and nutrition—using dietary intake and blood concentrations of micronutrients—with arsenic metabolism, as reflected in the proportions of InAS, MMA, and DMA in urine, in the first study that incorporated both dietary and micro­ nutrient data. Methods : We studied methylation patterns and nutritional factors in 405 persons who were selected from a cross­sectional survey of 7 ,638 people in an arsenic­exposed population in West Bengal, India. We assessed associations of urine creatinine and nutritional factors (19 dietary intake variables and 16 blood micronutrients) with arsenic metabolites in urine. r esult : Urinary creatinine had the strongest relationship with overall arsenic methylation to DMA. Those with the highest urinary creatinine concentrations had 7.2% more arsenic as DMA compared with those with low creatinine (p < 0.001). Animal fat intake had the strongest relation­ ship with MMA% (highest tertile animal fat intake had 2.3% more arsenic as MMA, p < 0.001). Low serum selenium and low folate were also associated with increased MMA%. c onclusion : Urine creatinine concentration was the strongest biological marker of arsenic methylation efficiency, and therefore should not be used to adjust for urine concentration in arsenic studies. hTe new finding that animal fat intake has a positive relationship with MMA% warrants further assessment in other studies. Increased MMA% was also associated, to a lesser extent, with low serum selenium and folate. k ey words : arsenic, creatinine, diet, India, methylation, micronutrients, West Bengal. Environ Health Perspect 119:1308–1313 (2011). http://dx.doi.org/10.1289/ehp.1003393 [Online 7 June 2011] to DMA(V) in a second methylation step then reduces toxicity. Epidemiological stud- ies have found evidence of increased risks of skin and bladder cancer among persons with higher proportions of MMA in their urine (Steinmaus et al. 2003). Therefore, factors that influence arsenic methylation may be important determinants of susceptibility to health effects resulting from exposure to InAs in drinking water. We were also interested in urine creatinine, because it is frequently used to adjust for the concentration of urine, something that would introduce bias if creati- nine were related to arsenic methylation. Methods Study participants. This study involved 405 participants selected from a study base of 7,683 individuals living in the South 24-Parganas district of West Bengal, India, who were identified in a 1995 through 1996 cross-sectional survey (Guha Mazumder et al. 1998). Participants had been selected for a case–control study of skin lesions caused by arsenic involving 192 cases and 213 controls (Haque et al. 2003), whose primary sources of drinking water contained < 500 µg/L InAs [n = 4,185 (2,160 females and 2,025 males)]. Cases had positive skin-lesion classifica- tions, with either hyperpigmentation (mot- tled, dark-brown pigmentation bilaterally distributed on the trunk), keratoses (diffuse thickening of palms or soles, with or with- out nodules), or both conditions at the time of the survey. Of the 7,683 individuals in the base population, we randomly selected controls from a subset of 4,185 individuals, with drinking water concentrations of arsenic < 500 µg/L; controls were matched to cases Address correspondence to A.H. Smith, Arsenic Health Effects Research Group, School of Public Health, University of California, Berkeley, 50 University Hall, MC 7360, Berkeley, CA 94720 USA. Telephone: (510) 843-1736. E-mail: ahsmith@ berkeley.edu The authors gratefully acknowledge the assistance of C. Alston in preparation of this paper. United This work was supported by National Institutes of Health grants P42-ES04705 and D43-TW000815 and Nations Children’s Fund SSA/ INDQ/2001/00003082-1. The authors declare they have no actual or potential competing financial interests. Received 28 December 2010; accepted 7 June 2011. ovlume 119 | number 9 | September 2011 • Environmental Health Perspectives Arsenic in drinking water is a major public health problem in the Bengal delta, affecting millions of people in the West Bengal state of India and adjoining areas in Bangladesh. In view of widespread poor nutrition prevalent in West Bengal and Bangladesh, it is important to identify dietary factors that might increase arse- nic toxicity or metabolism. We have already reported on the relationship of dietary constit- uents and blood micronutrients with arsenic- caused skin lesions in West Bengal (Chung et al. 2006; Mitra et al. 2004). Here, we report the findings concerning 19 dietary intake con- stituents, 16 blood micronutrients, and urine creatinine with arsenic methylation patterns in urine, in what we believe is the first com- prehensive joint assessment of dietary intake and micronutrients. For brevity, we refer to the dietary intake factors, along with the blood micronutrients, as “nutritional factors.” Nutritional factors may play important roles in methylation of inorganic arsenic (InAs), and the results of numerous studies have shown that methylation affects the toxicity of ingested InAs. In drinking water, InAs exists in trivalent [As(III)] and pentavalent [As(V)] forms. After ingestion, As(V) is reduced to 1308 As(III), followed by sequential methylation and reduction reaction steps resulting in for- mation of trivalent and pentavalent methylated arsenicals—first monomethyl acids [MMA(III) and MMA(V)] and subsequently trivalent and pentavalent dimethyl acids [DMA(III) and DMA(V), respectively. However, this process is incomplete, and populations show variations in the proportions of urinary InAs and the methylated arsenical compounds (Aposhian 1997; Concha et al. 2002; Drobna et al. 2004; Healy et al. 1997; Hopenhayn-Rich et al. 1996a, 1996b; Hopenhayn et al. 2003; Hughes 2002). I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f MMA(III) indicates that the first step of methylation that produces MMA should be considered an activation step. Laboratory studies have shown that the trivalent forms of MMA and DMA are much more toxic than the pentavalent forms, and in vitro evidence suggests that MMA(III), in particular, may be more toxic than trivalent InAs [InAs(III)] (Cullen and Reimer 1989; Lin et al. 1999, 2001; Mass et al. 2001; Petrick et al. 2000; Styblo et al. 1999, 2000; Styblo and Thomas 1997). The conversion of MMA(III)