Dose-responsive gene expression changes in juvenile and adult mummichogs (Fundulus heteroclitus) after arsenic exposure

Abstract

The present study investigated arsenic's effects on mummichogs (Fundulus heteroclitus), while also examining what role that gender or exposure age might play. Adult male and female mummichogs were exposed to 172 ppb, 575 ppb, or 1720 ppb arsenic as sodium arsenite for 10 days immediately prior to spawning. No differences were noted in the number or viability of eggs between the groups, but there was a significant increase in deformities in 1720 ppb arsenic exposure group. Total RNA from adult livers or 6-week old juveniles was used to probe custom macroarrays for changes in gene expression. In females, 3% of the genes were commonly differentially expressed in the 172 and 575 ppb exposure groups compared to controls. In the males, between 1.1 and 3% of the differentially expressed genes were in common between the exposure groups. Several genes, including apolipoprotein and serum amyloid precursor were commonly expressed in either a dose-responsive manner or were dose-specific, but consistent across genders. These patterns of regulation were confirmed by QPCR. These findings will provide us with a better understanding of the effects of dose, gender, and exposure age on the response to arsenic.

Fig. 1. Arsenic body burdens

Arsenic concentrations were measured in the adult females after the 10 day exposure. Data is the average ± standard deviation of 5 intact fish per group. Significant differences (*) were determined using Student's t-test (p<0.05).

Fig. 2. Gene expression profiles for males and female Fundulus exposed to different concentrations of arsenic

Hepatic gene expression was examined using the Fundulus cDNA array (GEO-GPL2535) and analyzed for differential expression using the CLEAR Test Method. The numbers of genes with significantly different expression (p≤0.05) are indicated, and those changed by more than one exposure are found in the overlapping areas. A. Dose-responsive changes in hepatic gene expression in male and female fish B. Gender-responsive changes in hepatic gene expression in male and female fish.

Fig. 2. Gene expression profiles for males and female Fundulus exposed to different concentrations of arsenic

Hepatic gene expression was examined using the Fundulus cDNA array (GEO-GPL2535) and analyzed for differential expression using the CLEAR Test Method. The numbers of genes with significantly different expression (p≤0.05) are indicated, and those changed by more than one exposure are found in the overlapping areas. A. Dose-responsive changes in hepatic gene expression in male and female fish B. Gender-responsive changes in hepatic gene expression in male and female fish.

Fig. 3. Percentage viability and morphological abnormalities following arsenic exposure

Adults were exposed to 172, 575 or 1,720ppb arsenic for 10 days prior to spawning. Eggs were collected for three days following arsenic exposure, and their viability, along with any developmental abnormalities observed after 7 days, was recorded. A. Viability and deformity percentages are expressed as the average + standard deviation for offspring from 4 –5 tanks, with statistical differences (*)determined by Mann-Whitney (p<0.05). B. Representative photographs from normal and deformed offspring. C. Morphological analysis of the vertebrae. The total size (area) for individual vertebrae were quantified using the LSM5 Pascal software. Values are the average area in square micrometers divided by the weight of the 6-week old fish. Significant differences (*) were determined using Student's t-test (p<0.05).

Fig. 4. Comparisons of gene expression changes between the arrays and quantitative real-time PCR (QPCR)

Expression of three genes from the arrays that were differentially expressed in almost all genders, ages, and exposure groups were verified by QPCR using specific primers (n=5 for each group). Array fold changes were plotted against QPCR fold changes to determine their directionality and magnitude. Genes examined were apolipoprotein, serum amyloid precursor, and an EST termed 9-7. The letter indicates females (F), males (M), or juveniles (J), while the number (172, 575, or 1,720) indicates the exposure group.