Grantee Research Project Results
1999 Progress Report: Mechanism of Salinity-Induced Toxicity of Aldicarb in Euryhaline Fish
EPA Grant Number: R826109Title: Mechanism of Salinity-Induced Toxicity of Aldicarb in Euryhaline Fish
Investigators: Schlenk, Daniel
Institution: University of California - Riverside
EPA Project Officer: Hahn, Intaek
Project Period: December 29, 1997 through December 28, 2000
Project Period Covered by this Report: December 29, 1998 through December 28, 1999
Project Amount: $263,149
RFA: Exploratory Research - Environmental Biology (1997) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
Certain agrichemicals are more toxic to euryhaline fish at high salinity. Mechanisms explaining this relationship are not known. Expression of a family of enzymes found in euryhaline fish, the flavin-containing monooxygenases (FMOs), which are known to bioactivate aldicarb, also are directly related to salinity. The project was designed to test the hypothesis that one or more forms of FMO, which are upregulated during high salinity are responsible for the bioactivation and subsequent enhanced toxicity of the thioether pesticide, aldicarb, in euryhaline fish. In Year 2, portions of each specific aim were carried out: (1) the effects of salinity on aldicarb uptake, and the interactions of aldicarb and its metabolites with acetylcholinesterase were examined in the euryhaline fish, the Japanese Medaka (Oryzias latipes); (2) the effect of salinity on In vitro aldicarb metabolism in medaka was examined and demonstrated a significant role of FMO in aldicarb activation; and (3) the effect of salinity on FMO expression and activity in medaka also was examined.Progress Summary:
Specific Aim 1: Examine the effect of salinity on aldicarb and its receptor cholinesterase, as well as uptake and elimination in medaka.- Toxicity. Each of these sub-aims has been accomplished. The 96h LC50s calculated were 0.48 with a 95 percent confidence interval of 0.33-0.68 ppm, 0.58 (0.45-0.76, 95 percent confidence limits), 0.50 (0.42-0.59, 95 percent confidence limits), and 0.13 (0.06-0.31 95 percent confidence limits), for adult males, females (8 weeks old), juveniles (34 weeks old), and larvae (48 h old), respectively. Salinity significantly enhanced the toxicity of aldicarb to both male and female medaka. In addition, the results revealed that females were more sensitive to the salinityinduced enhanced toxicity. Mortality percentage increased from 13 percent in males residing at 1.5 ppths salinity to 56 percent at 20 ppths, while toxicity significantly increased to 76 percent in the case of female medaka.
- Acetylcholinesterase (AChE) Inhibition. A time course characterization of the inhibition of AChE was conducted by exposing the fish to the 96h LC20, LC50, and LC90 concentrations following a 2-week acclimation period to the above-mentioned salinity regimens. Results showed that muscle AChE inhibition was directly correlated to the degree of toxicity, and that female AChE was significantly more sensitive to inhibition by aldicarb, especially at the high salinity regimens. In females residing at 20 ppths salinity, muscle AChE was inhibited 93.5 percent + 3.3 percent by 8 h of aldicarb exposure, while, in males, the maximum inhibition reached was 79.7 + 7.4 percent.
- Uptake and Elimination. Uptake and elimination of 14C aldicarb was measured in the total body of both genders and was unchanged by salinity (El-Alfy and Schlenk, 1998). There were no significant differences in elimination half-lives between the two genders.
In vitro Biotransformation. In vitro biotransformation was assessed in males and females and demonstrated enhanced activation of aldicarb to aldicarb sulfoxide in animals at higher salinity. Co-incubation with CO indicated sulfoxidation was not mediated by CYP and was nine-fold higher in the susceptible females (specifically gill tissues). Whole animal extracts were used to determine in vivo biotransformation with poor recovery of metabolites from the tissue matrix. Specific activity of the radiolabel did not allow the appropriate sensitivity needed for in vivo studies. It is proposed that future studies utilize canulated trout in which salinity also enhances toxicity.
Specific Aim 3: Characterize the effect of salinity on FMO expression in medaka.
- Effects of salinity on expression of FMO activity and FMO-like protein. Performed previously (Schlenk and El-Alfy, 1998). New antibodies provided from Dr. Allan Rettie from the University of Washington have identified a potential FMO3-like protein in the liver of medaka. Newer Anti-FMO1 antibodies also provided by Dr. Rettie recognize a single band, which appears to correlate with activity. Gender comparisons indicated higher levels of expression in the gill of females which correlated with enzyme activity, aldicarb bioactivation, and toxicity.
- Effects of osmoregulatory modulating compounds on FMO activity and FMO-like proteins in medaka. Adult Japanese medaka were separated by sex and exposed to 100 ppb estradiol or testosterone for 6 days followed by exposure to the 96h LC50 of aldicarb. The expression of FMO1 and FMO3 isoforms was monitored using anti-human antibodies. Testosterone significantly reduced FMO1 expression in male livers and gills. Testosterone did not affect the expression of FMOs in female tissues. Estradiol, on the other hand, significantly increased gill FMO1 expression in females. Aldicarb acts by the inhibition of the enzyme AChE, thus the effect of sex hormones on this enzyme pre- as well as post-aldicarb exposure also was examined. In both male and female medaka, testosterone counteracted the inhibitory effect of aldicarb on muscle AChE. Estradiol had similar effects in male fish but did not seem to counteract the AChE inhibition in females.
- Effects of osmoregulatory modulating compounds on toxicity. Estradiol and testosterone significantly lowered the toxicity of aldicarb to adult males. In females, testosterone caused significant reduction in aldicarb toxicity while estradiol significantly enhanced the toxicity. Measurement of brancial FMOs activity showed a direct correlation between the hormonal effect on FMOs activity in this tissue and the overall toxicity of aldicarb in both male and female medaka. In conclusion, estradiol and testosterone modulation of aldicarb toxicity in Japanese medaka seems to be mediated via alteration of gill FMOs activity as well as modulation of AChE activity.
The direct relationship between aldicarb sulfoxide formation, FMO activity and salinity is consistent with our proposed mechanism: salinity upregulates FMO in euryhaline fish, which leads to enhanced aldicarb sulfoxide formation that is a more potent cholinesterase inhibitor which enhances toxicity. In contrast to preliminary data that indicated down regulation of FMO by estradiol, sexually mature males appear to be more resistant to aldicarb toxicity than females. In addition, salinity enhanced toxicity was not observed in juvenile medaka, but was observed in larvae (which were the most sensitive life stage overall). Muscle AChE correlated well with acute toxicity in all groups examined and is consistent as a primary mechanism for aldicarb toxicity. The ramifications of these results suggest hazard assessments for ecological risk assessments should include a euryhaline species and toxicity studies should be carried out in a range of salinities.
Future Activities:
In the future, we will: (1) complete elimination studies using whole animal medaka and canulated rainbow trout models; (2) complete in vivo biotransformation studies either through extraction of depuration water of medaka, or using trout model; and (3) examine the effects of osmoregulatory hormones on FMO expression in rainbow trout. Preliminary molecular biology studies have indicated a FMO1-like gene in trout and medaka (see presentations above). Through isolation of the gene, a better understanding of its regulation by osmoregulatory hormones will be achieved.Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 11 publications | 3 publications in selected types | All 3 journal articles |
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El-Alfy AT, Grisle S, Schlenk D. Characterization of salinity-enhanced toxicity of aldicarb to Japanese medaka: Sexual and developmental differences. Environmental Toxicology and Chemistry 2001;20(9):2093-2098. |
R826109 (1999) R826109 (2000) |
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Supplemental Keywords:
susceptibility, metabolism, risk assessment, pesticides, estuary, runoff, gender, age., RFA, Scientific Discipline, Toxics, Ecosystem Protection/Environmental Exposure & Risk, Toxicology, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, pesticides, Ecological Effects - Environmental Exposure & Risk, Biochemistry, Ecology and Ecosystems, Ecological Risk Assessment, Ecological Indicators, ecological exposure, flavin containing monooxygenases (FMOs), hydrological, Japanese medaka, Euryhaline fish, salinity, Aldicarb, physiologically-based toxicokinetic models (PBTK), agrochemcial, aquatic ecosystemsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.