The involvement of several enzymes in methanol detoxification in Drosophila melanogaster adults

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Abstract

Methanol is among the most common short-chain alcohols in fermenting fruits, the natural food and oviposition sites of the fruit fly Drosophila melanogaster. Our previous results showed that cytochrome P450 monooxygenases (CYPs) were associated with methanol detoxification in the larvae. Catalases, alcohol dehydrogenases (ADHs), esterases (ESTs) and glutathione S-transferases (GSTs) were specifically inhibited by 3-amino-1,2,4-triazole (3-AT), 4-methylpyrazole (4-MP), triphenyl phosphate (TPP) and diethylmeleate (DEM), respectively. CYPs were inhibited by piperonyl butoxide (PBO) and 1-aminobenzotriazole (1-ABT). In the present paper, the involvements of these enzymes in methanol metabolism were investigated in female and male adults by determining the combination indices of methanol and their corresponding inhibitors. When PBO, 1-ABT, 3-AT, 4-MP and TPP were individually mixed with methanol, they exhibited significant synergism to the mortality of the adults after 72 h of dietary exposure. In contrast, the DEM and methanol mixture showed additive effects. Moreover, methanol exposure dramatically increased CYP activity and up-regulated mRNA expression levels of several Cyp genes. Bioassays using different strains revealed that the variation in ADH activity and RNAi-mediated knockdown of α-Est7 significantly changed LC50 values for methanol. These results suggest that CYPs, catalases, ADHs and ESTs are partially responsible for methanol elimination in adults. It seems that there are some differences in methanol metabolism between larvae and adults, but not between female and male adults.

Introduction

Methanol is well absorbed via ingestion, inhalation and skin exposure and has acute and chronic toxicities to animals (Cruzan, 2009, Sweeting et al., 2011). It is eliminated by both oxidative and non-oxidative pathways in several vertebrate species (Harris et al., 2003). In oxidative metabolism, methanol is converted into formaldehyde through one of at least three separate pathways. The first involves cytochrome P450 monooxygenases (CYP) such as CYP2E1 in humans (Coon and Koop, 1987, Wallage and Watterson, 2008). The second process is greatly dependent upon catalase (Cederbaum and Qureshi, 1982). The third pathway is catalyzed by cytosolic alcohol dehydrogenase (ADH I) via NAD+-dependent oxidation (Harris et al., 2003, MacAllister et al., 2011). Formaldehyde is further oxidized to produce formate by means of aldehyde dehydrogenase (ALDH II) in mitochondria, or by a series of reactions involving glutathione (GSH)-dependent formaldehyde dehydrogenase (ADH III) in the cytosol (Teng et al., 2001). Lastly, formate is eliminated by a tetrahydrofolate-dependent dehydrogenase to CO2 and water (Johlin et al., 1987, Harris et al., 2003). In non-oxidative metabolism, methanol is detoxified to form fatty acid methyl esters in vertebrates (Aleryani et al., 2005).

Plants produce and release considerable quantities of methanol, especially during early stages of leaf, flower and fruit development (Sharkey, 1996). When attacked by herbivore larvae, plants dramatically increase methanol production and release (Penuelas et al., 2005, von Dahl et al., 2006, Körner et al., 2009, Dorokhov et al., 2012a, Dorokhov et al., 2012b). Moreover, methanol is also a product of decaying organic material. In rotting cactus species, for example, methanol concentrations are from 0.9 to 33.3 mM (Starmer et al., 1986). In apples that have just started to decompose by fungus Sclerotinia fructigena, methanol concentration is about 39.3 μg/g. When the apple tissue is completely rotted, its content reaches up to 730 μg/g (Janzen, 1977). Therefore, it is possible that both herbivorous and saprophagous insects may suffer from methanol intoxication. However, scientific researches on methanol metabolism in insects are rare. Up to now, only two preliminary studies have been reported by our group. In the larvae of Asian corn borer Ostrinia furnacalis, ADH, CYP, catalase and esterase may participate in methanol elimination (Guo et al., 2010). In Drosophila melanogaster larvae, CYPs is indicated to be involved in methanol metabolism (Wang et al., 2012).

Some differences in metabolism of another alcohol, ethanol, have been found between Drosophila larvae and adults (Heinstra et al., 1983, Heinstra et al., 1989, Anderson and Barnett, 1991, Leal and Barbancho, 1992), or between males and females (Pfeiler and Markow, 2001, Pfeiler and Markow, 2003). In D. melanogaster larvae, more than 75% of the acetaldehyde is converted to acetate by ADH, even though both aldehyde dehydrogenase (ALDH) and ADH coexist (Heinstra et al., 1983, Heinstra et al., 1989). Conversely, acetaldehyde in adults is mainly oxidized into acetate by means of ALDH enzymes (Anderson and Barnett, 1991, Leal and Barbancho, 1992). In adult males of Drosophila pachea, a cactophilic species endemic to the Sonoran Desert of North America, ADH activity is substantially decreased 2 days after eclosion, and completely disappears by 4 days. In contrast, relatively high level of ADH activity persists throughout the adult life of females (Pfeiler and Markow, 2001, Pfeiler and Markow, 2003). It is expected, therefore, that there are some differences for methanol detoxification among various development stages and between different sexes in D. melanogaster.

3-Amino-1,2,4-triazole (3-AT), 4-methylpyrazole (4-MP), diethylmeleate (DEM) and triphenyl phosphate (TPP) are specific inhibitors of catalases, ADHs, glutathione S-transferases (GSTs) and esterases (ESTs), respectively. Piperonyl butoxide (PBO) was able to inhibit both CYPs and esterases (Moores et al., 2009), whereas 1-aminobenzotriazole (1-ABT) was a non-selective inhibitor to CYPs (Dierks et al., 1998). In the present paper, we focused on the adults. We tested the combined toxicities of the six enzyme inhibitors and dietary methanol to the mortality of the adults, measured CYP activity to p-nitroanisole (PNOD) and expression of a subset of Cyp genes after methanol exposure, and bioassayed methanol tolerance levels of several Drosophila stocks. Our results implied that CYPs, catalases, ADHs and ESTs were partially responsible for methanol detoxification in adults. It seems that there are some differences in methanol metabolism between larvae and adults, but not between female and male adults in D. melanogaster.

Section snippets

Experimental animals

The Canton-S (CS) and w1118 flies were used as control strains. The homozygote AdhFF, AdhSS and Adhn1 strains, UAS-α-Est7-dsRNA (y[1] sc[⁎] v[1]; P{y[+ t7.7] v[+ t1.8] = TRiP.HMS00955}attP2), UAS-EGFP-dsRNA (w[1118]; P{w[+ mC] = UAS-EGFP.dsRNA.R}142), transgenic flies, Act5C-Gal4, da-Gal4 and armadillo-Gal4 driver lines were obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN, USA).

The flies were raised on standard cornmeal/molasses/agar medium under controlled

LC50 values in male and female adults for the test chemicals

One day after treatment, methanol content in the flies feeding on diet contained methanol at the concentration of 39.5 mg/g was 6.7 times higher than in flies feeding on normal diet, indicating that methanol was absorbed by treated flies.

For both male and female D. melanogaster flies, LC50 values for methanol were similar to those for methanol plus acetone respectively based on overlapping 95% fiducial limits. These indicate that acetone exhibited little oral and fumigating toxicity to the flies

Discussion

Methanol is among the most common short-chain alcohols in the food and the oviposition substrates for Drosophila species that are adapted to survive in alcohol-rich niches. Our previous research implied that CYPs are involved in methanol elimination in the larvae (Wang et al., 2012). It is of interest to carry out further research to evaluate the detoxification activities of other metabolic enzymes such as ADHs, catalases and esterases, as well as CYPs, to methanol in D. melanogaster adults,

Acknowledgments

The research is supported by the National Basic Research Program of China (973 Program, No. 2010CB126200). We thank Drs. Z. Han and S. Dong of our laboratory for useful discussions during the course of this research.

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