Gut microbiome and function are altered for individuals living in high fluoride concentration areas in Pakistan.

BACKGROUND: Endemic fluorosis refers to the condition when individuals are exposed to excessive amounts of fluoride ion due to living in a region characterized by elevated levels of fluorine in the drinking water, food, and/or air. In Pakistan, a substantial proportion of the population is thereby affected, posing a public health concern. OBJECTIVES: Assessing how the gut microbiota and its metabolic profiles are impacted by chronic exposure to fluoride in drinking water (that caused Dental Fluorosis) as well as to perceive how this microbiota is connected to adverse health outcomes prevailing with fluoride exposure. METHODS: Drinking water (n=27) and biological samples (n=100) of blood, urine and feces were collected from 70 high fluoride exposed (with Dental Fluorosis) and 30 healthy control (without Dental Fluorosis) subjects. Water and urinary fluoride concentrations were determined. Serum/plasma biochemical testing was performed. Fecal DNA extraction, 16S rRNA analysis of microbial taxa, their predicted metabolic function and fecal short chain fatty acids (SCFAs) quantification were carried out. RESULTS: The study revealed that microbiota taxonomic shifts and their metabolic characterization had been linked to certain host clinical parameters under the chronic fluoride exposure. Some sets of genera showed strong specificity to water and urine fluoride concentrations, Relative Fat Mass index and SCFAs. The SCFAs response in fluoride-exposed samples was observed to be correlated with bacterial taxa that could contribute to adverse health effects. CONCLUSIONS: Microbial dysbiosis as a result of endemic fluorosis exhibits a structure that is associated with risk of metabolic deregulation and is implicated in various diseases. Our results may form the development of novel interventions and may have utility in diagnosis and monitoring.

Arsenic exposure induces urinary metabolome disruption in Pakistani male population.

Millions of people are at risk of consuming arsenic (As) contaminated drinking water in Pakistan. The current study aimed to investigate urinary arsenic species [iAsIII, iAsV, dimethylarsinic acid (DMA), methylarsonic acid (MMA)] and their potential toxicity biomarkers (based on urinary metabolome) in order to characterize the health effects in general adult male participants (n = 588) exposed to various levels of arsenic in different floodplain areas of Pakistan. The total urinary arsenic concentration (mean; 161 µg/L) of studied participants was lower and/or comparable than those values reported from other highly contaminated regions, but exceeded the Agency for Toxic Substances and Disease Registry (ATSDR) limits. For all the participants, the most excreted species was DMA accounting for 65% of the total arsenic, followed by MMA (20%) and iAs (16%). The percentage of MMA detected in this study was higher than those of previously reported data from other countries. These results suggested that studied population might have high risk of developing arsenic exposure related adverse health outcomes. Furthermore, random forest machine learning algorithm, partial correlation and binary logistic regression analysis were performed to screen the arsenic species-related urinary metabolites. A total of thirty-eight metabolites were extracted from 2776 metabolic features and identified as the potential arsenic toxicity biomarkers. The metabolites were mainly classified into xanthines, purines, and amino acids, which provided the clues linking the arsenic exposure with oxidative stress, one-carbon metabolism, purine metabolism, caffeine metabolism and hormone metabolism. These results would be helpful to develop early health warning system in context of arsenic exposure among the general populations of Pakistan.

Integrated Adsorption-Photodegradation of Organic Pollutants by Carbon Xerogel/Titania Composites.

Recent studies on the removal of pollutants via adsorption include the use of carbon-based adsorbents, due to their high porosity and large surface area; however, such materials lack photoactive properties. This study evaluates the synergistic effect of integrated mesoporous carbon xerogel (derived from resorcinol formaldehyde) and titanium dioxide (TiO2) for combined adsorption and photodegradation application. The complex formed between carbon xerogel and TiO2 phase was investigated through FTIR, proving the presence of a Ti-O-C chemical linkage. The physicochemical properties of the synthesised adsorbent-photocatalyst were probed using FESEM, BET analysis and UV-Vis analysis. The kinetics, equilibrium adsorption, effect of pH, and effect of adsorbent dosage were investigated. The expansion of the absorbance range to the visible range was verified, and the corresponding band gap evaluated. These properties enabled a visible light response when the system was exposed to visible light post adsorption. Hence, an assistive adsorption-photodegradation phenomenon was successfully executed. The adsorption performance exhibited 85% dye degradation which improved to 99% following photodegradation. Further experiments showed the reduction of microorganisms under visible light, where no microbial colonies were observed after treatment, indicating the potential application of these composite materials.

Monitoring and prediction of high fluoride concentrations in groundwater in Pakistan.

Concentrations of naturally occurring fluoride in groundwater exceeding the WHO guideline of 1.5 mg/L have been detected in many parts of Pakistan. This may lead to dental or skeletal fluorosis and thereby poses a potential threat to public health. Utilizing a total of 5483 fluoride concentrations, comprising 2160 new measurements as well as those from other sources, we have applied machine learning techniques to predict the probability of fluoride in groundwater in Pakistan exceeding 1.5 mg/L at a 250 m spatial resolution. Climate, soil, lithology, topography, and land cover parameters were identified as effective predictors of high fluoride concentrations in groundwater. Excellent model performance was observed in a random forest model that achieved an Area Under the Curve (AUC) of 0.92 on test data that were not used in modeling. The highest probabilities of high fluoride concentrations in groundwater are predicted in the Thar Desert, Sargodha Division, and scattered along the Sulaiman Mountains. Applying the model predictions to the population density and accounting for groundwater usage in both rural and urban areas, we estimate that about 13 million people may be at risk of fluorosis due to consuming groundwater with fluoride concentrations >1.5 mg/L in Pakistan, which corresponds to ~6% of the total population. Both the fluoride prediction map and the health risk map can be used as important decision-making tools for authorities and water resource managers in the identification and mitigation of groundwater fluoride contamination.

Super-Sensitive LC-MS Analyses of Exposure Biomarkers for Multiple Mycotoxins in a Rural Pakistan Population.

High levels of mycotoxin contamination have been reported in various food commodities in Pakistan, however, there has been no exposure assessment study using multiple mycotoxins' biomarkers. This study aimed to simultaneously assess the exposure to the five major mycotoxins: aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin B1 (FB1), ochratoxin A (OTA) and zearalenone (ZEN) in a Pakistani population using an integrated approach of human biomonitoring. Human urine samples (n = 292) were analyzed by a super-sensitive liquid-chromatography tandem mass spectrometry (LC-MS/MS) method. Rice and wheat were also collected and analyzed for mycotoxins by the LC-MS/MS method. Food consumption data were collected using a 24 h recall method. A high prevalence of urinary AFM1 (66%, mean ± SD 20.8 ± 41.3 pg/mL) and OTA (99%, 134.7 ± 312.0 pg/mL) were found, whilst urinary DON, FB1 and ZEN levels were low. The probable daily intake (PDI) derived from the urinary biomarkers revealed that 89% of the participants had exposure to OTA exceeding the established tolerable daily intake (TDI = 17 ng/kg bw/day). The average PDI of AFB1 for the studied population was 43 ng/kg bw/day, with rice as the main source of AFB1 exposure. In summary, exposure to AFB1 and OTA are of health concern and require further management.

Estimating the health burden of aflatoxin attributable stunting among children in low income countries of Africa.

Numerous population-based studies have documented high prevalence of aflatoxin associated childhood stunting in low income countries. We provide an estimate of the disease burden of aflatoxin related stunting using data from the four African countries. For this empirical analysis, we obtained blood aflatoxin albumin adduct biomarker based exposure data as measured using ELISA technique and anthropometric measurement data from surveys done over a 12-year period from 2001 to 2012 in four low income countries in Africa. We used these data to calculate population attributable risk (PAR), life time disease burden for children under five by comparing two groups of stunted children using both prevalence and incidence-based approaches. We combined prevalence estimates with a disability weight, measuring childhood stunting and co-occurrence of stunting-underweight to produce years lived with disability. Using a previously reported mortality, years of life lost were estimated. We used probabilistic analysis to model these associations to estimate the disability-adjusted life-years (DALYs), and compared these with those given by the Institute for Health Metrics and Evaluation's Global Burden of Disease (GBD) 2016 study. The PAR increased from 3 to 36% for aflatoxin-related stunting and 14-50% for co-occurrence of stunting and underweight. Using prevalence-based approach, children with aflatoxin related stunting resulted in 48,965.20 (95% uncertainty interval (UI): 45,868.75-52,207.53) DALYs per 100,000 individuals. Children with co-occurrence of stunting and underweight due to exposure to aflatoxin resulted in 40,703.41 (95% UI: 38,041.57-43,517.89) DALYs per 100,000 individuals. Uncertainty analysis revealed that reducing aflatoxin exposure in high exposure areas upto non-detectable levels could save the stunting DALYs up to 50%. The burden of childhood all causes stunting is greater in countries with higher aflatoxin exposure such as Benin. In high exposure areas, these results might help guide research protocols and prioritisation efforts and focus aflatoxin exposure reduction. HEFCE Global Challenge Research Fund Aflatoxin project.

Biomonitoring of Aflatoxin B1 and Deoxynivalenol in a Rural Pakistan Population Using Ultra-Sensitive LC-MS/MS Method.

There are limited data on exposure to mycotoxins in Pakistan. Here, we measured exposure to deoxynivalenol (DON), a common contaminant of wheat, and aflatoxin B1 (AFB1), a known contaminant of rice, using biomarkers of exposure. Wheat (n = 195) and rice (n = 62) samples were analyzed for AFB1 and DON levels, and the corresponding urinary biomarkers were analyzed in urine samples from a rural population (n = 264, aged 4-80 years, male 58%) using ultra-sensitive liquid chromatography-tandem mass spectrometry. AFB1 was detected in 66% of rice (5.04 ± 11.94 µg/kg) and 3% of wheat samples. AFM1 (hydroxylated form of AFB1)was detected in 69% of urine samples, mean 0.023 ± 0.048 ng/mL and DON was detected in 20% of urine samples, mean 0.170 ± 0.129 ng/mL. The maximum probable daily intake for DON derived from the urinary biomarker was 59.8 ng/kg b.w./day, which is below the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives' tolerable daily intake (1000 ng/kg b.w./day). However, for aflatoxin, the derived margin of exposure (MoE) of (13.2) was well below the safe MoE (10,000) suggested by the European Food Safety Authority. The calculated aflatoxin-associated cancer risk of 0.514/105 individuals/year suggests that measures should be taken to reduce the AFB1 contamination in food, particularly rice, in Pakistan.

Urinary profiles of selected metals and arsenic and their exposure pathway analysis in four large floodplains of Pakistan.

In context of fragile geological conditions and rapid urbanization, element exposure via dietary (food, water) and non-dietary (dust, soil) routes into human population at different land use settings is a major concern in the Indus floodplains (FPs) of Pakistan. In current study, several important trace elements including arsenic (As), chromium (Cr), manganese (Mn), cobalt (Co), cadmium (Cd), nickel (Ni), copper (Cu) and lead (Pb) were analyzed in the paired human urine, food, water and dust samples collected from main FPs of Pakistan. Daily intake estimation and regression analysis were used to evaluate the relationships between internal exposure, exposure routes of studied trace elements and different land use settings. High concentrations of urinary As, Cr, Cu, Mn, and Cd were detected in the general male population of the studied floodplains (FPs). Moreover, the levels (µg/L) of urinary As increased gradually from FP1 (12.8), FP2 (18), FP3 (61) to FP4 (71). Regression analysis showed that As contaminated water was correlated with elevated urinary As concentrations in FP3 and FP4, and water Cr and Mn was significantly associated with urinary Cr and Mn concentrations in FP2. Moreover, the associations of food Mn and urinary Mn were found in FP1. Over all, cumulative estimated daily intake (EDI) values from water, dust and food from all the flood plains showed that Mn had the highest values (6.6, 9.2, 14.4 µg/kg/day) followed by water As (1.98 µg/kg/day), dust Cu (1.5 µg/kg/day) and Pb (1.7 µg/kg/day). Studied floodplains were moderately to highly polluted in terms of studied trace elements (As, Cr, Cu, Mn, and Cd) contamination especially in FP3 and FP4. The results will contribute to improve the knowledge and information on current exposure of Pakistani male adults to the different contaminants.

Environmental exposure pathway analysis of trace elements and autism risk in Pakistani children population.

The pursuit of industrialization and urbanization in developing countries disrupt the fragile environment, resulting in biogeochemical extra-emission of the trace elements into human inhabitance causing serious health concerns. We aimed to determine the associations between Autism spectrum disorder (ASD) risk and exposure to trace elements (As, Zn, Ni, Pb, Hg, Cu, Cd, and Co), associations between the internal doses and environmental sources of these elements were also assessed. Genetic susceptibility to toxins was assessed through GSTT1 and GSTM1 null polymorphism analysis. Our results showed that lower BMI in children was significantly associated with ASD (p < 0.05, AOR = 0.86; 95% CI: 0.76, 0.98). As was significantly higher in both hair (p < 0.01, AOR = 18.29; 95% CI: 1.98, 169) and urine (p < 0.01, AOR = 1.04; 95% CI: 1.01, 1.06) samples from children with ASD; urinary Hg (p < 0.05, AOR = 2.90; 95% CI: 1.39, 6.07) and Pb (p < 0.05, AOR = 1.95; 95% CI: 1.01, 3.77) were also positively associated with ASD. Regarding the genetic susceptibility, Cu was significantly associated with GSTM1 positive genotype (p < 0.05, AOR = 1.05; 95% CI: 1.00, 1.10). Children inhabiting the urban areas exposed to significantly higher levels of studied trace elements. The Estimated Daily Intake (EDI) values highlighted that the different land use settings resulted in children's source specific exposure to studied trace elements. The exposure pathway analysis showed that the distal factors of land-use settings associated with children increased exposure risk for most of the investigated elements, noticeably As, Pb and Hg associated with ASD prevalence.

Assessment of arsenic species in human hair, toenail and urine and their association with water and staple food.

Arsenic intake from household drinking/cooking water and food may represent a significant exposure pathway to induce cancer and non-cancer health effects. This study is based on the human biomonitoring of 395 volunteers from 223 households with private water sources located in rural Punjab, Pakistan. This work has shown the relative contribution of water and staple food to arsenic intake and accumulation by multiple biological matrix measurements of inorganic and organic arsenic species, while accounting for potential confounders such as age, gender, occupation, and exposure duration of the study population. Multi-variable linear regression showed a strong significant relationship between total arsenic (tAs) intake from water and concentrations of tAs, inorganic arsenic (iAs), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) in urine and toenail samples. tAs intake from staple food (rice and wheat) also showed a strong significant relationship with hair tAs and iAs. The sole impact of staple food intake on biomarkers was assessed and a significant correlation was found with all of the urinary arsenic metabolites. Toenail was found to be the most valuable biomarker of past exposure to inorganic and organic arsenic species of dietary and metabolic origin.

Arsenic species in wheat, raw and cooked rice: Exposure and associated health implications.

Arsenic concentrations above 10µgL-1 were previously found in 89% of ground water sources in six villages of Pakistan. The present study has ascertained the health risks associated with exposure to total arsenic (tAs) and its species in most frequently consumed foods. Inorganic arsenic (iAs) concentrations were found to be 92.5±41.88µgkg-1, 79.21±76.42µgkg-1, and 116.38±51.38µgkg-1 for raw rice, cooked rice and wheat respectively. The mean tAs concentrations were 47.47±30.72µgkg-1, 71.65±74.7µgkg-1, 105±61.47µgkg-1. Wheat is therefore demonstrated to be a significant source of arsenic exposure. Dimethylarsinic acid was the main organic species detected in rice, whilst monomethylarsonic acid was only found at trace levels. Total daily intake of iAs exceeded the provisional tolerable daily intake of 2.1µgkg-1day-1 body weight in 74% of study participants due to concurrent intake from water (94%), wheat (5%) and raw rice (1%). A significant association between tAs in cooked rice and cooking water resulted in tAs intake 43% higher in cooked rice compared to raw rice. The study suggests that arsenic intake from food, particularly from wheat consumption, holds particular significance where iAs is relatively low in water. Chronic health risks were found to be significantly higher from wheat intake than rice, whilst the risk in terms of acute effects was below the USEPA's limit of 1.0. Children were at significantly higher health risk than adults due to iAs exposure from rice and/or wheat. The dietary exposure of participants to tAs was attributable to staple food intake with ground water iAs <10µgL-1, however the preliminary advisory level (200µgkg-1) was achievable with rice consumption of ≤200gday-1 and compliance with ≤10µgL-1 iAs in drinking water. Although the daily iAs intake from food was lower than total water intake, the potential health risk from exposure to arsenic and its species still exists and requires exposure control measures.

The effect of association between inefficient arsenic methylation capacity and demographic characteristics on the risk of skin lesions.

This study was conducted in rural Pakistan to assess the dose-response relationship between skin lesions and arsenic exposure and their variation by demographic characteristics. The study included 398 participants (66 participants with skin lesions and 332 without) residing in six previously unstudied villages exposed to ground water arsenic in the range of <1 to 3090µgL-1. The skin lesions identification process involved interview and physical examinations of participants followed by confirmation by a physician according to UNICEF criteria. Urinary inorganic arsenic (iAs), total arsenic (tAs), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were analysed to determine methylation capacity, methylation efficiency and the dose-response relationship with skin lesions. Study participants with skin lesions were found to be exposed to arsenic >10µgL-1 with a daily arsenic intake of 3.23±3.75mgday-1 from household ground water sources for an exposure duration of 10-20years. The participants with skin lesions compared to those without skin lesions showed higher levels of urinary iAs (133.40±242.48 vs. 44.24±86.48µgg-1Cr), MMA (106.38±135.04 vs. 35.43±39.97µgg-1Cr), MMA% (15.26±6.31 vs.12.11±4.68) and lower levels of DMA% (66.99±13.59 vs. 73.39±10.44) and secondary methylation index (SMI) (0.81±0.11 vs. 0.86±0.07). Study participants carrying a lower methylation capacity characterized by higher MMA% (OR 5.06, 95% CI: 2.09-12.27), lower DMA% (OR 0.64, 95% CI: 0.33-1.26), primary methylation index (PMI) (OR 0.56, 95% CI: 0.28-1.12) and SMI (OR 0.43, 95% CI: 0.21-0.88) had a significantly higher risk of skin lesions compared to their corresponding references after adjusting for occupation categories. The findings confirmed that inefficient arsenic methylation capacity was significantly associated with increased skin lesion risks and the effect might be modified by labour intensive occupations.

Refinement of arsenic attributable health risks in rural Pakistan using population specific dietary intake values.

BACKGROUND: Previous risk assessment studies have often utilised generic consumption or intake values when evaluating ingestion exposure pathways. If these values do not accurately reflect the country or scenario in question, the resulting risk assessment will not provide a meaningful representation of cancer risks in that particular country/scenario. OBJECTIVES: This study sought to determine water and food intake parameters for one region in South Asia, rural Pakistan, and assess the role population specific intake parameters play in cancer risk assessment. METHODS: A questionnaire was developed to collect data on sociodemographic features and 24-h water and food consumption patterns from a rural community. The impact of dietary differences on cancer susceptibility linked to arsenic exposure was evaluated by calculating cancer risks using the data collected in the current study against standard water and food intake levels for the USA, Europe and Asia. A probabilistic cancer risk was performed for each set of intake values of this study. RESULTS: Average daily total water intake based on drinking direct plain water and indirect water from food and beverages was found to be 3.5Lday-1 (95% CI: 3.38, 3.57) exceeding the US Environmental Protection Agency's default (2.5Lday-1) and World Health Organization's recommended intake value (2Lday-1). Average daily rice intake (469gday-1) was found to be lower than in India and Bangladesh whereas wheat intake (402gday-1) was higher than intake reported for USA, Europe and Asian sub-regions. Consequently, arsenic-associated cumulative cancer risks determined for daily water intake was found to be 17 chances in children of 3-6years (95% CI: 0.0014, 0.0017), 14 in children of age 6-16years (95% CI: 0.001, 0.0011) and 6 in adults of 16-67years (95% CI: 0.0006, 0.0006) in a population size of 10,000. This is higher than the risks estimated using the US Environmental Protection Agency and World Health Organization's default recommended water intake levels. Rice intake data showed early life cumulative cancer risks of 15 chances in 10,000 for children of 3-6years (95% CI: 0.0012, 0.0015), 14 in children of 6-16years (95% CI: 0.0011, 0.0014) and later life risk of 8 adults (95% CI: 0.0008, 0.0008) in a population of 10,000. This is lower than the cancer risks in countries with higher rice intake and elevated arsenic levels (Bangladesh and India). Cumulative cancer risk from arsenic exposure showed the relative risk contribution from total water to be 51%, from rice to be 44% and 5% from wheat intake. CONCLUSIONS: The study demonstrates the need to use population specific dietary information for risk assessment and risk management studies. Probabilistic risk assessment concluded the importance of dietary intake in estimating cancer risk, along with arsenic concentrations in water or food and age of exposed rural population.

Human exposure assessment of different arsenic species in household water sources in a high risk arsenic area.

Understanding arsenic speciation in water is important for managing the potential health risks associated with chronic arsenic exposure. Most arsenic monitoring studies to date have only measured total arsenic, with few looking at arsenic species. This study assessed 228 ground water sources in six unstudied villages in Pakistan for total, inorganic and organic arsenic species using ion chromatography inductively coupled plasma collision reaction cell mass spectrometry. The concentration levels approached 3090µgL-1 (95% CI, 130.31, 253.06) for total arsenic with a median of 57.55µgL-1, 3430µgL-1 (median=52) for arsenate (As+5) and 100µgL-1 (median=0.37) for arsenite (As+3). Exceedance of the WHO provisional guideline value for arsenic in drinking water (10µgL-1) occurred in 89% of water sources. Arsenic was present mainly as arsenate (As+5). Average daily intake of total arsenic for 398 residents living in the sampled houses was found up to 236.51µgkg-1day-1. This exposure estimate has indicated that 63% of rural residents exceeded the World Health Organization's provisional tolerable daily intake (PTDI) of 2.1µgkg-1day-1 body weight. Average daily intake of As+5 was found to be 15.63µgkg-1day-1 (95% CI, 5.53, 25.73) for children ≤16 and 15.07µgkg-1day-1 (95% CI, 10.33, 18.02) for adults. A mean daily intake of 0.09µgkg-1day-1 was determined for As+3 for children and 0.26µgkg-1day-1 for adults. Organic arsenic species such as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and Arsenobetaine (AsB) were found to be below their method detection limits (MDLs).