Effects of Embryonic Arsenic Exposure on Killifish (Fundulus heteroclitus) Growth, Feeding Behavior, and Intestinal Morphology and Cell Types

Arsenic is found as a contaminant of drinking water, rice, and other crops. Epidemiological studies have shown that embryonic exposure to arsenic can cause changes in behavior and reductions in growth, but the mechanisms for these effects are not well understood. So, we were interested in examining potential mechanisms by which arsenic could be affecting growth. Additionally, while many studies have looked at higher levels of arsenic exposure, we wanted to focus on environmentally-relevant levels to see if these concentrations could have lasting consequences on growth, even after the exposure had ended. Killifish (Fundulus heteroclitus) were used as the model organism for this investigation for two reasons. First, they produce a large number of eggs, which can increase statistical power when observing affects over multiple time points. Second, earlier studies have shown effects on developmental processes at arsenic levels similar to human exposures. In rodent or zebrafish models, investigators typically need to use arsenic concentrations that are 100X higher to see similar effects. Thus, killifish were exposed to 0, 10, 50, and 200ppb arsenic (as sodium arsenite) as embryos, and after hatching were reared in clean water until adulthood at 28 weeks. The study was designed to represent a full embryonic/fetal arsenic exposure in utero, and then to examine whether effects persisted, worsened, or resolved into early adulthood. We found that growth, assessed by condition factor (weight/length³), was significantly reduced by 24% in the 200 ppb embryonic exposure groups at 8 weeks, with a dose dependent decrease in the 10 and 50 ppb groups. These trends persisted up to 28 weeks, although variability was much higher. As we had seen similar reductions in growth in a previous embryonic arsenic exposure study that used higher arsenic concentrations, we therefore investigated three potential mechanisms responsible for the growth reduction. First, we analyzed feeding behavior, as it has been found to correlate to amount of nutrient intake. Embryonic arsenic exposure did indeed reduce the percentage of fish initially responding to food and increased the amount of time it took for the fish to start their response, particularly at the 28 week time period. So, one possibility is that arsenic reduces activity or alters olfaction, thus reducing their response to food. The second mechanism examined was whether embryonic arsenic exposure altered the morphology of the intestine, or altered several specific cell types needed for nutrient uptake. There was a slight, but statistically significant reduction in intestinal villus height at 16 weeks, this change did not persist. Intestinal enterocytes and Goblet cell number, as measured by immunohistochemistry, did not change with arsenic concentration or time. However, the number of PCNA-positive intestinal cells, indicating cell proliferation, was reduced in a dose-response manner at all sampling time points. This may indicate that embryonic arsenic exposure permanently altered the ability of intestinal stem cells to proliferate. The third possibility we examined was whether embryonic arsenic exposure altered the expression of skeletal and hepatic insulin like growth factor (IGF-1), its receptor (IGFR-1) on skeletal muscle cells, and its associated binding proteins (IGFBP-1 & -5) in the muscle and liver. We hypothesized that changes in their levels might alter growth and muscle body weight, since epidemiological studies have found an inverse relationship between arsenic and IGF-1 in plasma levels, which correlate to reductions in birth weight. Reductions of hepatic IGF1 and IGFBP-1 are highly correlated with condition factor reductions in the 8 week old fish. However, by 28 weeks, hepatic IGF-1 and IGFBP-1 still remain tightly correlated, but are actually increased in a statistically significant, dose-response manner. This might be a compensatory response to potentially making up for any growth deficits seen in earlier stages. Overall, the results from this study show that embryonic-only arsenic exposure can alter growth factor expression, such as hepatic IGF-1, which correlates with a reduction in condition factor during an essential growth period such as the juvenile stage. As the fish reach sexual maturity, it appears that by increasing levels of IGF-1 and restoring a consistent intestinal environment, they are able to compensate for early growth deficits after embryonic exposure to lower levels of arsenic