## 4 Discussion
### Inorganic As Methylation in the Liver of the Experimental Animals  
The methylation of inorganic As species in mammals occurs through a specific enzymatic process. The pathway involves, first, the reduction of inorganic As(V) to inorganic As(III), which is then followed by oxidative methylation processes to produce monomethylarsonic acid--As MMA(V) and dimethylarsinic acid--As DMA(V) metabolites [80]. The methylation of inorganic As species is widely known as a detoxification process since the methylated As species MMA(V) and DMA(V) are less toxic than the inorganic forms [84; 85]. However, this assertion has been questioned since the intermediate MMA(III) and DMA(III) metabolites are known to be more toxic As species [85; 86]. In the literature, for easy understanding, the assessment of inorganic As methylation capacity in mammals has been categorised into primary methylation (first phase of methylation) and secondary methylation (second phase of methylation) [87]. The interpretation is that the primary methylation represents the proportion of MMA transformed from the total amount of As species present, and the proportion of DMA derived from the total As species indicates secondary methylation [88]. In this study, we calculated the % iAs (the sum of AsIII and AsV), the % MMA, and the % DMA to represent their relative proportions per the total sum of detectable As species (AsIII + AsV + MMA + DMA) in percentages. The % iAs represents the relative proportion of the inorganic As that is not methylated/metabolised in the liver. % MMA shows the efficiency of the first phase of the methylation process, and % DMA represents the efficiency of the second phase of the methylation process [59]. From the results, % iAs among the animal groups was in the order As group-As + CBZ group > CO group > CBZ group. For the % MMA and % DMA, the sequence was CBZ group > CO group > As + CBZ group-As group. The CBZ group had the lowest % iAs, suggesting that it has the least unmetabolised inorganic As species in the liver after the termination of the experiment. In other words, this means the CBZ group was the most efficient or the fastest in transforming the inorganic As species to MMA and DMA metabolites. This is corroborated by the highest % MMA and % DMA in the CBZ group compared to the other animal groups, indicating the highest primary and secondary methylation capacities. The As and As + CBZ groups had high and comparable % iAs, suggesting these groups had comparably high amounts of unmetabolised/unmethylated inorganic As. This also implied the inefficient capacities of these animal groups to metabolise or methylate inorganic As species to MMA and DMA. This is confirmed by the lowest % MMA and % DMA recorded for these groups. Recent findings have shown that the predominant pathway for inorganic metabolism is the enzymatic process catalysed by arsenite methyltransferase enzyme (As3 MT) leading to methylated As species of MMA(V) and DMA(V) [59; 80; 83]. [PERSON] et al. [59], observed that As3 MT expression correlated negatively with % iAs and positively with % DMA in the liver. These previous findings suggest that an efficient As3 MT catalysed methylation reaction leads to lower % iAs and higher % DMA. In other words, lower unmetabolised inorganic As species and higher secondary methylation capacity of inorganic As. According to [PERSON] et al. [80] and [PERSON] et al. [83], the liver has high levels of GSH, especially in mice. Thus, this leads to high quantities of As(III)-(GS)\({}_{3}\) complexes, which serve as the substrate for As3 MT. However, at high % iAs, As3 MT becomes saturated from the accumulated As(III) and this may cause the inhibition of the enzyme, leading to a decrease in the capacity to convert inorganic As to the methylated metabolites [59; 89].