International Journal of Toxicology and Toxicity Assessment

Stability of monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) in human urineand mouse tissues

Uttam K Chowdhury — 2023-04-16

Urine samples were collected from 75 subjects in theLagunera area of Mexico. There were four groups, based on totalarsenic concentrations in their drinking water(9-100μg/L).After collection,the samples were immediately put in a portable iceboxcontaining dry ice, and they were kept frozen while beingtransported to the University of Arizona, Tucson where theywerestored at -70°C before analysis.Arsenic metabolites,including MMA(III) and DMA(III),were measured in urine samples by usingHPLC-ICP-MS.

The average percentage of MMA(III) in urine samples of arsenic exposed people in the Lagunera area of Mexico were 0.44%, 0.26%, N.D. (Not Detectable), and 0.20% of total arsenic for the groups 4, 1, 3, and 2 where arsenic concentrations in their drinking water were 9, 17.5, 52, and 100 (µg/L), respectively.This small percentage of MMA(III) were detected only in 14urine samples (~18%)out of total 75 samples. DMA(III) was not measured in any of these urine samples (n=75) but measured DMA(V) in allsamples.The highest percentage of arsenic metabolites was DMA(V) (61% to 74%) of the total arsenic in urine. It indicates thatmost of the MMA(III) methylated to DMA(V) in tissues,butlesspercentageor most of the DMA(V) may be reducedto DMA(III) and due toits instability,itcould be immediately oxidized to DMA(V)in tissues again or in urine after collection. These findingssuggest that the +5-oxidation state of arsenic metabolite, DMA(V), could be themost dominant methylated arsenic metabolite in humans’ urine of arsenic exposed population.

In animal tissues,the +3-oxidation state of arsenic metabolites,MMA(III) and DMA(III),were measured in mice tissues after administering a single intra-muscular dose of sodium arsenate(4.16 mg As/kg body weight).Liver, kidneys, urinary bladder tissue, lungs, testes, and heart were removed at the following times, 0, 0.5, 1, 2, 4, 8, and 12 h.The tissues were homogenized at 4°C (cold room),and the homogenized samples were extracted immediately. After extraction, I measured the arsenic species as soon as possible by using HPLC-ICP-MS(Chowdhury, et al., 2006).

The concentration of the very toxic MMA(III) was significantly higher than that of MMA(V) in all of the tissues tested (Liver, kidneys, urinary bladder tissue, lungs, testes, and heart). At 2 h, after injection, the highest concentration of MMA(III) was in the kidneys.Compared with the other species of arsenic, MMA(V)concentrations were the lowest in all tissues examined.

On the other hand, the concentration of DMA(V) was higher than DMA(III) in the liver,kidneys,urinary bladder tissue, lungs, and testes at all times. DMA(V) accumulated at a higher concentration in the urinary bladder tissue and lungs, but the concentration of DMA(III) was the highest in the urinary bladder tissue, followed by the kidneys, lungs, heart, testes, and liver. In all of the tissues, both DMA(V) and DMA(III) were highest at 4 h for the WT (wildtype) mice. The concentration of DMA(III) was significantly higher in the urinary bladder tissue than in other tissues (p<0.05), except in the kidneys of the mice.

The results indicate that MMA(V) reduced to MMA(III) faster, comparing to DMA(V) to DMA(III). It could be that most of the MMA(V) reduced to MMA(III) and methylated to DMA(V), but less/most of the MMA(III) converted to DMA(V)↔ DMA(III), but DMA(III) may be very instable and oxidized to DMA(V) very quickly. This might have been because the DMA(III) would be more instable in the tissues, comparing to MMA(III), and oxidized faster, compared to MMA(III). Also, it could be suitable for the bonding of MMA(III) to tissue components or proteins,compared to DMA(III).

These results also indicate that due to the instability and faster oxidation of DMA(III),therewas no detectable level of DMA(III), but low level of MMA(III) was measuredin some urine samples due to its more stability and bonding capacity with proteins,compared to DMA(III). Both MMA(III) and DMA(III) were detected in tissues but not in urines, and these reports have suggested that tissue levels of MMA(III) and DMA(III) are both more relevant and less susceptible to oxidative artifacts than urine samples.

In conclusion, the distributionof arsenic metabolites in urine was supported with the distribution of these species in mouse tissues.The trivalent MMA(III) compound could be themost dominant,very toxic arsenic metabolite in humans’ tissues fora short period of time (e.g., MMA(III) was highest at 2 h in mouse tissuesafter injectionof sodium arsenate) but not in urine because it methylated to DMA(V) in tissues, and some percentage of MMA(III) could also be oxidized to MMA(V) again. On the other hand, DMA(V), instead of DMA(III), would be another dominant arsenic metabolite in tissues as well as in urine because we found the final metabolites of arsenic in urine where most of the arsenic metabolite was DMA(V).Thestability/instability of MMA(III) and DMA(III)may depend on biological environment, genetic variability, and other factors.However,more experiments are needed to understand the mechanism of inorganic arsenic biotransformationand stability or instability of highly toxic arsenic metabolites, MMA(III) and DMA(III).

Arsenic Health Impacts to the People in Bangladesh

Uttam K Chowdhurya — 2023-10-07

In a period from 1995 to 2000, we had collected and analyzed 33,092 hand tube well water samples from four different/principal geomorphological regions (Hill tract, Table Land, Flood Plain, and Deltaic reason) i.e., from all 64 districts of Bangladesh and found arsenic in 60 districts that were above the WHO recommended value in drinking water (10 μg/L) and 50 districts that were above the maximum permissible limit, 50 μg/L.

In our study for 6 years in Bangladesh, the survey for identification of arsenical patients was conducted by our group with a medical team in 261 villages of 80 police stations under 33 districts out of 50 where contamination of groundwater with arsenic is above 50 μg/L. We could not identify people suffering from arsenical skin lesions who drunk water below 100 μg/L of arsenic.

During this survey, arsenic patients were identified in 222 villages of 69 police stations under 31 out of 33 districts. The number of people we examined including children was 18840, and 3725 people were identified with arsenical skin lesions. We had registered 1885 males, 1542 females, and 298 children out of the total 3725 patients, having arsenical dermal lesions, such as: melanosis, leucomelanosis, keratosis, hyperkeratosis, dorsal, non-petting oedema, gangrene, cancer, etc. If children are included, then 19.77% (n=3725) have arsenical dermal lesions, and for separately adults and children, these are 24.52% and 6.13%, respectively. Buccal mucus membrane melanosis (MMM) on tongue, gums, lips, etc. was also found. Rough dry skin often with palpable nodules (spotted keratosis) on dorsal of hand, feet, and legs are the symptoms seen in severe cases.

Other symptoms are sometimes found (1) conjunctional congestion and (2) non-petting swelling (solid oedema) of feet. Complications like liver enlargement (hepatomegaly), spleen enlargement (splenomegaly) and fluid in abdomen are seen in severe cases. Squamous cell carcinoma, basal cell carcinoma, Bowen's disease, carcinoma affecting lung, uterus, bladder, genitourinary tract, or other sites are often seen in advanced neglected cases suffering for many years. During our preliminary field survey in Bangladesh, we could identify 25 carcinoma /gangrene patients out of 3725 patients. In addition to the above symptoms, we observe some common problems in arsenic patients with arsenical skin lesions such as intolerance to sunlight, burning sensation on whole body, weakness, and respiratory problem.

In children, our last 6 years of field experience in Bangladesh show that normally children under 11 years of age do not show arsenical skin manifestations. However, we have observed a few exceptions when (1) the arsenic content in water consumed by children is very high (1000 μg/L) and (2) the arsenic content is not very high (around 500 μg/L), but the children get poor nutrition. The youngest arsenic patient was a child (age 18 months) with melanosis (+) and keratosis (++) [spotted on palm and sole] who was found in Payerpur village under Madaripur Sadar police station in Madaripur district. While discussing with his mother, I came to know that the child used to drink a very high quantity of water (2-3 liters per day) from childhood.

So far, we had examined 4,864 children, below 11 years of age, drinking arsenic contaminated water from the arsenic affected villages in Bangladesh and out of that arsenical skin lesions registered from 298 children (6.12%) whereas in adults it is 24.47%. Normally, arsenical skin lesions observed in children are diffuse melanosis and spotted melanosis. Keratosis on the palm and sole are not common in children. We have not found children suffering from+++ stage of melanosis and keratosis (we used mild+, moderate to high++, severe+++). Also, we have not found any child patient during last 6 years in Bangladesh, below 11 years of age, with non-pitting oedema, gangrene, Bowens, dorsal, or cancer.

The members of the under-poverty line family versus the rich family: In addition to common arsenical symptoms, we also observed dorsal (15%), chronic bronchitis (30%), and vitamin deficiency among the under-poverty line family members, but such type of symptoms were not observed to the members in rich family. Children at the age around 11 years in that family had no arsenical skin lesions. The arsenic concentration in drinking water of the tube well was 570 μg/L. But six children out of seven, around 11 years of age, were found with arsenical skin lesions in the under-poverty line family. The youngest victim was 8 years old, and the arsenic concentration in drinking water being used by this family was 690 μg/L.

Our field experience in Bangladesh, we have observed that among the adults suffering from diffuse melanosis and light spotted melanosis can recover after drinking safe water, eating nutritious food, and taking vitamins. Normally diffused melanosis disappears easily after drinking safe water, and light keratosis (+) may also disappear. But if keratosis is appreciably visible (++), drinking safe water and eating nutritious food may reduce it, but it may not disappear. In arsenic patients with moderate to severe (++ and +++) keratosis, the appearance of keratosis does not stop even after drinking safe water over a long period of time and even when hair, nail, and skin scales contain safe levels of arsenic.

We have further observed that children recover from diffuse melanosis (blackening of color) and light spotted melanosis (+) quickly if they use safe water, have better nutrition, and eat vitamins. Mild keratosis (+) also disappears, but the children having moderate to high spotted melanosis (++) and spotted keratosis (++), even after drinking safe water and nutritious food, do not recover completely. We had found diffuse melanosis in children disappear, and those who had spotted melanosis (+) and keratosis (+) are no longer showing skin lesions. Those who had ++ spotted melanosis and ++ spotted keratosis could not get rid of their skin lesions, and spotted melanosis is replaced by Leucomelanosis, and keratosis is less. However, the children are still complaining about their weaknesses, breathing problems, and suffering from cough and cold.

Finally, if it is accepted that children are at a higher risk due to arsenic exposure, then the future of the next generation of Bangladesh living in arsenic affected villages may be grim as above 84% and 89% of the children's hair and nail contain arsenic above toxic (hair) or normal level (nail), respectively. Therefore, it is very important to monitor the concentration of arsenic in their drinking water even though they are drinking arsenic free deep tube well water now.