Microsomal oxidation of fenthion to the oxon and sulfoxide was observed in liver and gills but not in olfactory tissues (). All metabolites were stable throughout the 60 min incubation with no observed hydrolysis of sulfoxides or fenoxon observed (data not shown). When liver microsomes from FA fish were incubated with fenthion, fenoxon production was lower compared to total production of fenthion sulfoxide (). In gills, fenoxon and fenthion sulfoxide production were up to 2.2-fold lower compared to liver (P<0.01). In olfactory tissues, no sulfoxidation of fenthion was observed. In all tissues, NADPH-independent and NADPH-dependent cleavage of fenthion was observed (). NADPH-independent production of MMTP from fenthion was higher in gills and olfactory tissues than in liver (P<0.01). NADPH-dependent cleavage of fenthion was significantly higher in olfactory tissues than in liver and gills (P<0.01).
Biotransformation of fenthion and related metabolites in microsomal fractions isolated from liver, gills and olfactory tissues in freshwater-acclimated rainbow trout (Oncorhynchus mykiss)
When microsomes were incubated with fenoxon (), total sulfoxidation to fenoxon sulfoxide was higher in liver and gills when compared to sulfoxidation of fenthion (up to 1.8-fold). This activity was also higher in liver compared to gills. Sulfoxidation was not observed in olfactory tissues. As observed for fenthion, NADPH-independent and dependent cleavage of fenoxon was detected. Higher NADPH-independent MMTP production from fenoxon was observed in gills. In olfactory tissues the NADPH-dependent activity was 1.7-fold and 2.2-fold higher compared to liver and gill tissues (P<0.01), respectively.
When each fenthion sulfoxide enantiomer was used as a substrate (S- or R-), minimal production of the corresponding fenoxon sulfoxide was observed in microsomal fractions from liver and gills (see ). The rate of fenoxon sulfoxide formation was lower than the oxidation of fenthion or fenoxon but still 2 to 3-fold higher in liver than in gills. There were no significant stereoselective differences in fenoxon sulfoxide production from the individual sulfoxide enantiomers.
In terms of stereoselective sulfoxidation of fenthion, the ratios of each enantiomer formed in liver and gill microsomes from FA fish favoured the formation of the S-fenthion sulfoxide. The observed ratio in both tissues was 65% S-fenthion sulfoxide and 35% R-fenthion sulfoxide. When microsomes isolated from both tissues were incubated with fenoxon, the observed ratio towards its fenoxon sulfoxides was different from the ratio for fenthion (74% S-fenoxon sulfoxide and 26% R-fenoxon sulfoxide; P<0.05).
As a positive control, biotransformation of fenthion and its metabolites was evaluated using recombinant human FMO1. When incubated with 100 μM fenthion, only R-fenthion sulfoxide production was observed (204.2±34.6 pmol/min/mg protein). When fenoxon was used as substrate, the R-fenoxon sulfoxide was the only metabolite produced and the rate of conversion to fenoxon sulfoxide was significantly higher (293.6±15.4 pmol/min/mg protein) than the conversion from fenthion to fenthion sulfoxide. Fenoxon sulfoxides were not produced from fenthion sulfoxide.
NADPH-independent or dependent cleavage of fenthion or related metabolites was not observed in cytosolic incubations isolated from liver, gills and olfactory rosettes, suggesting that no cytosolic enzymes (i.e. cytosolic esterases) were involved in detoxification pathways of this pesticide (data not shown).
The kinetics of fenthion metabolism was characterized in liver microsomes from FA fish and HA fish. As shown in , conversion to different metabolites indicated varied catalytic efficiencies. NADPH-independent MMTP production showed the highest apparent Km (292.0±66.6 μM), and the lowest Vmax. The apparent Km for fenthion sulfoxide production was 1.8 times higher than that for fenoxon production. In FA fish, higher catalytic efficiency was observed for the formation of NADPH-dependent MMTP followed by fenoxon production. However, in HA fish, a dramatic shift was observed to the formation of sulfoxides with efficiency of S-sulfoxide formation being approximately 3-fold higher than the R-sulfoxide. The efficiency of R-sulfoxide formation was 2-fold greater than fenoxon.
Enzymatic kinetic characterization of fenthion metabolites in microsomal fractions isolated from liver from freshwater- and hypersaline-acclimated rainbow trout (Oncorhynchus mykiss) and incubated with fenthion
Hypersaline conditions significantly increased total fenthion biotransformation by liver microsomes in rainbow trout (). Rates of fenoxon production were enhanced more than 1.5-fold in HA fish. The rate of fenthion sulfoxide production (S- and R-) was also enhanced nearly 3-fold in HA fish relative to FA fish. The formation of fenoxon sulfoxides (S- and R-) from fenthion was observed in liver microsomes from HA fish but not from FA fish. The NADPH-dependent production of MMTP was reduced up to 7-fold in liver microsomes of HA fish when compared to FA fish (P<0.01). In contrast, hypersaline conditions failed to alter the NADPH-independent cleavage of fenthion. Incubations with fenoxon, produced significantly higher rates of sulfoxide formation in HA fish (up to 2.4-fold and 2.8-fold for S- and R-fenoxon sulfoxide, respectively). Also, the production of S- and R-fenoxon sulfoxides from fenthion sulfoxide increased with hypersalinity. When liver microsomes from HA fish were incubated with fenoxon, the NADPH-dependent production of MMTP was significantly reduced (up to 14-fold) compared with FA fish (P<0.01), and the NADPH-independent activity remained unchanged.
Effects of hypersaline conditions on the in vitro biotransformation of fenthion in microsomes of liver from rainbow trout (Oncorhynchus mykiss)
In microsomal fractions isolated from gills of HA fish incubated with fenthion, fenoxon production remained unchanged compared to freshwater-treated fish, and S- and R-fenthion sulfoxidation was significantly reduced by 1.8-fold and 2-fold, respectively (P<0.01) (). The same trend was observed when gill microsomes from HA fish were incubated with fenoxon with S- and R-fenoxon sulfoxide production significantly reduced 2-fold and 2.2-fold, respectively (P<0.01). The respective production of S- and R-fenoxon sulfoxides from S- and R-fenthion sulfoxides was also reduced by hypersaline conditions to levels below detection (<0.3 pmol/min/mg protein). As in liver microsomal fractions, NADPH-independent production of MMTP remained unchanged in HA fish, but the NADPH-dependent formation of MMTP was significantly enhanced (P<0.01).
Effects of hypersaline conditions on the in vitro biotransformation of fenthion in microsomes of gills from rainbow trout (Oncorhynchus mykiss)
In olfactory tissues, sulfoxidation of fenthion and related metabolites was not detected in HA fish (data not shown). MMTP was produced through NADPH-independent and NADPH-dependent pathways from fenthion and fenoxon, but was unaffected by hypersaline exposure (P>0.05).
In terms of stereoselective sulfoxidation of fenthion, the synthesis ratios of each enantiomer remained unchanged (65% and 35%, S-fenthion sulfoxide and R-fenthion sulfoxide, respectively) in microsomes from liver and gills of HA fish relative to FA fish. In addition, hypersaline conditions failed to alter the stereoselectivity of fenoxon sulfoxidation in either liver or gill.
In liver microsomes, fenoxon formation was significantly reduced by co-incubation with ketoconazole in FA fish and HA fish, 67% and 60%, respectively (). Ketoconazole (500 μM) also inhibited testosterone hydroxylase below detection (<0.2 pmol/min/mg protein; data not shown). Water treatments did not cause fish to alter stereoisomer production. Ketoconazole failed to alter fenthion sulfoxide enantiomeric ratios when reactions were performed with microsomes isolated from FA or HA fish. Co-incubation with methimazole failed to alter fenoxon formation, but reduced total sulfoxide formation 28% and 35% in FA and HA fish, respectively. In contrast to ketoconazole, enantiomeric ratios were significantly altered from 65% S-fenthion sulfoxide to 75% S-fenthion sulfoxide specifically through the reduction of the R-sulfoxide in HA fish. Ratios were unchanged in FA fish. NADPH-independent and NADPH-dependent MMTP production was unaltered by exposure to ketoconazole or methimazole.
Table 5 The effects of the FMO inhibitor (methimazole, 500 μM) and CYP inhibitor (ketoconazole, 500 μM) on the biotransformation of fenthion in microsomal fractions isolated from liver from freshwater- and hypersaline-acclimated rainbow trout (more ...)
In all three tissues from FA fish, 100 μM TEPP completely inhibited the NADPH-independent cleavage of fenthion to MMTP (<0.3 pmol/min/mg protein) (). NADPH-dependent MMTP production was not altered at this TEPP concentration. However, co-incubation with 500 μM of TEPP led to a range of 46.4–57.3% inhibition of NADPH-dependent hydrolysis in microsomes of each tissue. Co-incubation with 1 mM TEPP reduced NADPH-dependent MMTP production to levels below detection all three tissues ().
MMTP [3-methyl-4-(methylthio)-phenol] formation from fenthion and TEPP (tetraethylpyrophosphate) inhibition in microsomal fractions isolated from liver, gills and olfactory tissues in freshwater-acclimated rainbow trout (Oncorhynchus mykiss)
Immunoblots of microsomes from liver showed that CYP1A levels were reduced 2-fold in HA fish (P<0.01) ( and ). CYP2K1 and CYP2M1 content remained unchanged in liver microsomes from animals from both exposure groups. Expression of hepatic CYP3A27 was significantly increased (P<0.01) in HA fish. The formation of 6β-hydroxytestosterone and 16β-hydroxytestosterone from testosterone was also increased (up to 2.6-fold and up to 1.5-fold, respectively) in liver microsomes from HA fish ().
Fig. 3 CYP1A, CYP2K1, CYP2M1 and CYP3A27 protein determined by Western blot in microsomal fraction isolated from liver from freshwater- and hypersaline-acclimated rainbow trout (Oncorhynchus mykiss). Data expressed as mean±SD (n=4–6 individuals (more ...)
Fig. 4 Western blots of CYP1A1 (A), CYP2K1 (B), CYP2M1 (C) and CYP3A27 (D)-like proteins in liver microsomal fractions from freshwater- (FAF) and hypersaline (HAF)-acclimated rainbow trout (Oncorhynchus mykiss). Protein load: 40 μg/lane. Molecular weight (more ...)
Fig. 5 Testosterone hydroxylase activity (CYP3A27-related) in microsomal fractions isolated from liver in freshwater- and hypersaline-acclimated rainbow trout (Oncorhynchus mykiss). Data expressed as mean±SD (n=8 individuals with 3 replicates for each (more ...)