Major Biotransformation Products Research Articles

Mechanism of β-blocker biodegradation by wastewater microorganisms.

The recalcitrant β-blockers have been widely detected in aquatic environments up to several hundred μg/L, which are major contributors to β1 antagonistic activities in wastewater. Their biodegradation mechanisms remain obscure, hindering the development of efficient removal techniques. This study constructed the biodegradation pathways for three typical β-blockers, namely atenolol, metoprolol, and propranolol, assessed the toxicity of their major biotransformation products, and identified the key enzyme catalyzing the O-dealkylation reaction leading to pollutant mineralization. Atenolol and metoprolol degradation was more efficient than that of propranolol by activated sludge, producing metoprolol acid (MTPA) as a major intermediate. Hydrogenophaga sp. YM1 isolated from activated sludge possess the α-ketoglutarate dependent dioxygenase (TfdA) responsible for O-dealkylation of MTPA and propranolol, producing 4-hydroxyphenylacetic acid (4-HPA) that can be further degraded and ultimately enters the TCA cycle. The role of TfdA was verified by proteomics, enzyme stimulation/inhibition tests, and gene knockout experiments. Molecular docking suggests its different interactions with MTPA and propranolol. Acetate facilitated the degradation of β-blockers efficiently. The results may shed light on enhanced biological removals of broader β-blockers and their transformation products in the environment.

Anammox enrichment culture has unexpected capabilities to biotransform azole contaminants of emerging concern

Azoles are contaminants of emerging concern. They have a ubiquitous presence in the environment due to their wide variety of uses. This study investigated the fate of two commonly occurring azole compounds in an anammox enrichment culture. The results showed that 1H-pyrazole (PA) and 1H-1,2,4-triazole (TA) were biotransformed yielding major biotransformation products, 3-amino-1H-pyrazole and 3-amino-1H-1,2,4-triazole, respectively. Nitrate and glucose greatly stimulated the biotransformation. Under optimized conditions, 80.7% of PA and 16.4% of TA were biotransformed in an incubation period of 6 days. High molar product yield of 84.5% and 83.6% was observed per mole of PA and TA biotransformed, respectively. This novel and selective biotransformation constitutes the first report on the microbial biotransformation of PA and is amongst the very few reports on the biotransformation of TA. This study also provides evidence that anammox enrichments have unexpected capabilities to biotransform organic contaminants of emerging concern.

Comparison of Transcriptomics Changes Induced by TCS and MTCS Exposure in Human Hepatoma HepG2 Cells.

Triclosan (TCS) has been a widely used antibacterial agent in medical and personal care products in the last few decades. Methyl TCS (MTCS) is the major biotransformation product of TCS through replacement of the hydroxyl group with methoxy. Previous studies revealed that MTCS showed reduced toxicity but enhanced environmental persistence, when compared with TCS. Till date, the toxicological molecular mechanisms of TCS and MTCS remain to be clarified. This study aimed to investigate the transcriptomic changes in HepG2 cells induced by TCS and MTCS using microarray chips and to identify key target genes and related signal pathways. The microarray data showed that there were 1664 and 7144 differentially expressed genes (DEGs) in TCS- and MTCS-treated groups, respectively. Gene ontology (GO) enrichment and Kyoto Encyclopedia of genes and genomes (KEGG) analysis revealed that TCS and MTCS induced overlapping as well as distinct transcriptome signatures in HepG2 cells. Both TCS and MTCS could result in various biological responses in HepG2 cells mainly responding to biosynthetic and metabolic processes but probably through different regulatory pathways. Among the selected 50 GO terms, 9 GO terms belonging to the cellular component category were only enriched in the MTCS group, which are mainly participating in the regulation of cellular organelle’s function. KEGG analysis showed that 19 and 59 pathway terms were separately enriched in TCS and MTCS groups, with only seven identical pathways. The selected 10 TCS-specific signal pathways are mainly involved in cell proliferation and apoptosis, while the selected 10 MTCS-specific pathways mainly take part in the regulation of protein synthesis and modification. The overall data suggested that MTCS induced more enriched DEGs, GO terms, and pathway terms than TCS. In conclusion, compared with TCS, MTCS presents lower polarity and stronger lipophilicity, enabling MTCS to cause more extensive transcriptomic changes in HepG2 cells, activate differentiated signal pathways, and finally lead to differences in biological responses.

Construction of the Heterologous Laccase Producer Aspergillus nidulans lac№4 (argB–) and Its Application for the Progesterone Transformation

A novel Aspergillus nidulans lac№4 (argB–) strain, a producer of recombinant laccase A from basidiomycete Trametes hirsuta 072, was obtained. This strain was used for the biocatalytic transformation of progesterone (PG). The major biotransformation products are 11α-hydroxy-PG, 11α-acetoxy-PG and 6β, 11α-dihydroxy-PG. Macronet MN-200 sorbent was used to study the adsorption kinetics of PG, and the main biotransformation products from the transformation medium. The solid-phase extraction of steroids without preliminary removal of the mycelium was shown to be efficient.

The In Vitro Biotransformation of the Fusion Protein Tetranectin-Apolipoprotein A1

As more and more protein biotherapeutics enter the drug discovery pipelines, there is an increasing interest in tools for mechanistic drug metabolism investigations of biologics in order to identify and prioritize the most promising candidates. Understanding or even predicting the in vivo clearance of biologics and to support translational pharmacokinetic modeling activities is essential, however there is a lack of effective and validated in vitro cellular tools. Although different mechanisms have to be adressed in the context of biologics disposition, the scope is not comparable to the nowadays widely established tools for early characterization of small molecule disposition. Here, we describe a biotransformation study of the fusion protein tetranectin apolipoprotein A1 by cellular systems. The in vivo biotransformation of tetranectin apolipoprotein A1 has been described previously, and the same major biotransformation product could also be detected in vitro, by a targeted and highly sensitive detection method based on chymotrypsin digest. In addition, the protease responsible for the formation of this biotransformation product could be elucidated to be DPP4. To our knowledge, this is one of the first reports of an in vitro biotransformation study by cells of a therapeutic protein.

Enhanced emerging pharmaceuticals removal in wastewater after biotreatment by a low-pressure UVA/FeIII-EDDS/H2O2 process under neutral pH conditions

The widespread occurrence of pharmaceutically active compounds (PhACs) in aquatic environment has become a worldwide concern. This work investigated the removal of four representative emerging PhACs, including carbamazepine (CBZ), crotamiton (CTMT), N,N-diethyl-meta-toluamide (DEET) and ibuprofen (IBP) in two internal circulation biological aerated filters (IcBAFs). Results showed that DEET (>95%) and IBP (>80%) were removed efficiently in the IcBAFs, whereas CTMT (<30%) and CBZ (<5%) were poorly removed due to their recalcitrant characteristics. The mechanism for DEET and IBP removal in the IcBAFs was biodegradation and autotrophic microbes were responsible for their degradation. The major biotransformation products of IBP included hydroxy-IBP and 1-(4-isobutylphenyl)ethanol, while one major biodegradation product of DEET was identified as N,N-diethylbenzamide. In addition, the removal of residual PhACs in IcBAF effluents by various advanced oxidation processes (AOPs) were evaluated in either batch or continuous-flow mode. The results showed that the low-pressure UVA/FeIII-EDDS/H2O2 process was very effective for the treatment of residual PhACs in IcBAF effluents under neutral pH conditions, e.g., resulting in more than 90% degradation of the PhACs in continuous-flow mode. The impacts of photo-Fenton reagents doses (i.e., FeIII-EDDS dose, H2O2 dose and the EDDS:FeIII molar ratio), as well as the UV light sources (UVA/UVC) and intensities on the residual PhACs removal were evaluated in detail. The LC-QTOF-MS analysis revealed that not only the target PhACs but also their biodegradation byproducts were efficiently degraded by the low-pressure UVA/FeIII-EDDS/H2O2 process, suggesting that this process had great potential for the treatment of residual PhACs in wastewater after biotreatment.

Biotransformation of macrolide antibiotics using enriched activated sludge culture: Kinetics, transformation routes and ecotoxicological evaluation

The biotransformation of three prominent macrolide antibiotics (azithromycin, clarithromycin and erythromycin) by an activated sludge culture, which was adapted to high concentrations of azithromycin (10 mg/L) was investigated. The study included determination of removal kinetics of the parent compounds, identification of their major biotransformation products (TPs) and assessment of ecotoxicological effects of biotransformation. The chemical analyses were performed by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry, which enabled a tentative identification of TPs formed during the experiments. The ecotoxicological evaluation included two end-points, residual antibiotic activity and toxicity to freshwater algae. The enriched activated sludge culture was capable of degrading all studied macrolide compounds with high removal efficiencies (>99%) of the parent compounds at elevated concentrations (10 mg/L). The elimination of all three macrolide antibiotics was associated with the formation of different TPs, including several novel compounds previously unreported in the literature. Some of the TPs were rather abundant and contributed significantly to the overall mass balance at the end of the biodegradation experiments. Biodegradation of all investigated macrolides was associated with a pronounced reduction of the residual antibiotic activity and algal toxicity, indicating a rather positive ecotoxicological outcome of the biotransformation processes achieved by the enriched sludge culture.

Microbial detoxification of carvedilol, a β-adrenergic antagonist, by the filamentous fungus Cunninghamella echinulata

Beta adrenergic antagonists like carvedilol are typical environmental pollutants detected in wastewater and surface water. Human metabolism of carvedilol is well investigated, while its environmental fates are still unknown. In recent years, there have been appearing reports on high toxicity of β-blockers toward aquatic organisms. In this paper the ability of the filamentous fungus C. echinulata to eliminate the β-blocker has been described for the first time. An 83% loss of carvedilol was observed after 120 h incubation of the tested fungus with the compound, where hydroxylated carvedilol metabolites were identified as the major biotransformation products. Carvedilol degradation by C. echinulata was proceeded by hydroxylation and conjugation reactions similar to its mammalian metabolism. Glucose conjugate was found in the fungi cultures, whereas glucuronide conjugates were detected in mammals. The impact of carvedilol on the functionality of fungal cells was also evaluated. A 2-fold decrease in the PC/PE ratio was noticed in the C. echinulata cell membrane after the exposition to carvedilol compared to control mycelium incubated without the β-blocker. The change can denote perturbation of fungal cell membrane integration by carvedilol. Moreover, 2.8-fold lower toxicity of postcultures supernatants toward D. magna were shown in contrast to abiotic control.

In vitro antimalarial studies of novel artemisinin biotransformed products and its derivatives

Biotransformation of antimalarial drug artemisinin by fungi Rhizopus stolonifer afforded three sesquiterpenoid derivatives. The transformed products were 1α-hydroxyartemisinin (3), 3.0%, a new compound, 10β-hydroxyartemisinin, 54.5% (4) and deoxyartemisinin (2) in 9% yield. The fungus expressed high-metabolism activity (66.5%). The chemical structures of the compounds were elucidated by 1D, 2D NMR spectrometry and mass spectral data. The major compound 10β-hydroxyartemisinin (4) was chemically converted to five new derivatives 5–9. All the compounds 3–9 were subjected for in vitro anti-malarial activity. 10β-Hydroxy-12β-arteether (8), IC50 at 18.29nM was found to be 10 times better active than its precursor 4 (184.56nM) and equipotent antimalarial with natural drug artemisinin whereas the α-derivative 9 is 3 times better than 4 under in vitro conditions. Therefore, the major biotransformation product 4 can be exploited for further modification into new clinically potent molecules. The results show the versatility of microbial-catalyzed biotransformations leading to the introduction of a hydroxyl group at tertiary position in artemisinin in derivative (3).

High-resolution mass spectrometric investigation of the phase I and II metabolites of finasteride in pig plasma, urine and bile

1. The metabolite profile of the 5α-reductase type II inhibitor finasteride has been studied in pig plasma, urine and bile using high-resolution mass spectrometry. The porcine biotransformation products were compared to those formed by human liver microsomes and to literature data of recently identified human in vivo metabolites. The objective of this study was to gain further evidence for the validity of using pigs for advanced, invasive drug–drug interaction studies that are not possible to perform in humans.2. The use of high-resolution mass spectrometry with accurate mass measurements enabled identification of the metabolites by calculation of their elemental compositions as well as their fragmentation patterns.3. There was an excellent match between the porcine and human metabolic profiles, corroborating the pig as a model of human drug metabolism. The glucuronides of the two recently described human hydroxylated metabolites MX and MY and the carboxylated metabolite M3 were identified as the major biotransformation products of finasteride in pig urine and bile.4. Furthermore, the CYP enzymes involved in the formation of the hydroxylated metabolites were characterized. Human recombinant CYP3A4 could produce the two major hydroxylated metabolites MX and MY, whereas human recombinant CYP2D6 formed MY only.

On the use of a high-redox potential laccase as an alternative for the transformation of non-steroidal anti-inflammatory drugs (NSAIDs)

The release of pharmaceutical compounds to the environment, such as non-steroidal anti-inflammatory drugs (NSAIDs), implies a great concern because of its negative effects, e.g. potential cytotoxicity to liver and kidney of animal species. In the present study, a high-redox potential laccase is proposed as an alternative system for the biotransformation of selected NSAIDs: naproxen (NPX) and diclofenac (DCF). The effects of pH and the use of synthetic and natural mediators (1-hydroxybenzotriazole (HBT) and syringaldehyde (SA), respectively) were evaluated with the aim of optimizing the removal of NSAIDs by laccase. Results proved high removal yields for NPX (70–94%) after 24 h in the presence of HBT at acidic and neutral pH, while DCF was completely transformed at pH 4 within shorter periods, from 30 min to 4 h, both in the absence or presence of mediators. The operation at a higher pH implied the addition of mediators to remove DCF, with values of 50 and 98% for SA and HBT, respectively. Kinetic parameters of the transformation reactions and laccase inactivation were estimated and compared for the different experimental conditions. Besides, the identification of the major biotransformation products of DCF was attempted: decarboxylated compounds were detected by gas chromatography-mass spectrometry and corresponding reaction pathways were proposed. Moreover, laccase-catalyzed treatment was demonstrated to significantly improve the aerobic biodegradability of the medium containing DCF in comparison with untreated solution, suggesting that DCF transformation products are less toxic than the parent compound.

Transformation of xanthohumol by Aspergillus ochraceus

Microbial transformation of xanthohumol isolated from agro-residue (spent hops), by Aspergillus ochraceus was investigated. A new aurone, (Z)-2″-(2‴-hydroxyisopropyl)-dihydrofurano[4″,5″:6,7]-3',4'-dihydroxy-4-methoxyaurone, was obtained as a main transformation product. Three minor metabolites were identified as 2″-(2‴-hydroxyisopropyl)-dihydrofurano[4″,5″:3',4']-2',4-dihydroxy-6'-methoxychalcone, (2S,2″S)-2″-(2‴-hydroxyisopropyl)-dihydrofurano[4″,5″:7,8]-4'-hydroxy-5-methoxyflavanone and (2S,2″R)-2″-(2‴-hydroxyisopropyl)-dihydrofurano[4″,5″:7,8]-4'-hydroxy-5-methoxyflavanone. Their structures were elucidated on the basis of spectroscopic evidences. The antioxidant properties of xanthohumol and its metabolites were investigated using the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. The major biotransformation product, was 8.6-fold stronger antioxidant than xanthohumol and 2.3-fold than ascorbic acid.

Combination of biotransformation by P450 BM3 mutants with on-line post-column bioaffinity and mass spectrometric profiling as a novel strategy to diversify and characterize p38α kinase inhibitors

Mutants of bacterial cytochrome P450 BM3 from Bacillus megaterium have gained increasing interest to support drug metabolism studies by producing human relevant biotransformation products and/or to support drug discovery by producing libraries of new chemical entities based on initial lead compounds. In this study, a library of 33 P450 BM3 mutants was used to diversify TAK-715, a representative (highly lipophilic) inhibitor of p38α mitogen-activated protein kinase (p38α). To simultaneously determine the individual bioaffinity and identity of the different products, an analytical high-resolution screening approach was used, based on post-column on-line bioaffinity profiling with parallel mass spectrometric identification. The screening of the product mixtures produced demonstrated that introducing mutations in the active site of the P450 BM3 resulted in different product profiles. Several P450 BM3 mutants were mimicking the metabolic profile obtained by human liver microsomes. The major biotransformation products of TAK-715 could be produced with the most active P450 BM3 mutant in sufficient amounts to enable further structure elucidation by 1H-NMR and quantification of their p38α affinity.

INVITED REVIEW: Efficacy, metabolism, and toxic responses to chlorate salts in food and laboratory animals1

For over 100 yr, scientists have explored uses of sodium chlorate in agricultural applications. Sodium chlorate is a strong oxidizer, and thus can be very hazardous when not handled accordingly. Nevertheless, late 19th century agriculturists and scientists attempted to exploit the chemical properties of sodium chlorate as an herbicide and food preservative. It is the herbicidal utility that led to subsequent use of sodium chlorate in the agricultural industry since then. However, in 2000, USDA-ARS scientists proposed a new and targeted use of sodium chlorate against enterobacteria in food animal production. Specifically, when orally dosed in to cattle (Bos taurus), swine (Sus scrofa), broilers (Gallus gallus), turkeys (Meleagris gallopavo), and sheep (Ovis aries), chlorate reduced the fecal shedding of common enteropathogens of the Enterobacteriaceae family. Subsequent to this discovery, the efficacy of chlorate salts has been demonstrated in numerous production classes within species. Doses of sodium chlorate as low as 30 mg/kg BW, but typically 50 to 150 mg/kg BW, have been used to demonstrate efficacy against pathogens. Single or short-duration (<3 d) exposures to oral chlorate at concentrations < 150 mg/kg BW have not produced acute toxicity or clinical signs (labored breathing, methemoglobinemia) in food animals. In all species studied to date, the major biotransformation product of chlorate is chloride ion; chlorite is not present in tissues or excreta of chlorate dosed animals. Chlorate is rapidly eliminated in ruminants and nonruminants, primarily in urine; likewise, residual chlorate in tissues depletes rapidly. Application of any new chemical entity to food animal production carries with it a responsibility to understand adverse reactions that intended and nonintended exposures may have in target and (or) nontarget animals and an understanding of the pathways of elimination that occur after exposure. Therefore, the purpose of this review is to summarize the published data regarding the efficacy, metabolism, and toxicology of chlorate salts in target (livestock) and nontarget species.

A sensitive and robust method for the determination of alkylphenol polyethoxylates and their carboxylic acids and their transformation in a trickling filter wastewater treatment plant

This paper presents a method for the determination of alkylphenols, alkylphenol polyethoxylates (APEO) and alkylphenol ethoxycarboxylates (APEC) in the aqueous and particulate phase of wastewater samples. Quantification was achieved by liquid chromatography-tandem mass spectrometry. The sensitivity of the method is demonstrated by low detection limits, in the dissolved phase 1.2–9.6 ng l −1 for alkylphenol, AP 1–3EO and APEC and 0.1–4.1 ng l −1 for longer chain alkylphenol polyethoxylates. The method detection limit for particulate phase samples ranged from 6 to 60 ng g −1 for AP, AP 1–3EO and APEC; with the longer chain APEO being from 0.5 to 20 ng g −1. Matrix effects were noted in complex matrix rich samples. There was a distinct change in the distribution of alkylphenol ethoxylates during biological treatment of the wastewater, with the major biotransformation products observed being carboxylated derivatives at concentrations of up to 1768 ng l −1. Shorter chain APEO were present in higher proportions in the suspended solids, due to their higher affinity to particulate matter compared to the long-chain oligomers.

Biotransformasi (−)-Epigalokatekin-3-O-galat Menjadi (−)-2R,3S-Dihidromirisetin oleh Fungi Endofit Diaporthe sp. Isolat E dari Tumbuhan Teh

Endophytic fungi have been reported possess an interesting ability to mimic their host plant metabolites. Several fungi also show their specific capability to biotransform the chemical constituents of the host plant. The endophytic fungus Diaporthe sp. E isolate obtained from young stem of a tea plant ( Camellia sinensis ) show their unique capability to biotransform (−)-epigallocatechin-3- O -gallate [(−)-EGCG] into a major product in glucose-peptone-yeast extract medium that incubated under dark condition at 27 °C for 48 h. The major biotransformation product were isolated and purified through column chromatography techniques using Sephadex LH-20 and silica gel. The chemical structure of the major product were elucidated as (−)-2 R ,3 S -dihydromyricetin based on their IR, FAB-MS, 1D- and 2D-NMR spectra.

Disposition and metabolic profiling of [14C]-Decabromodiphenyl ether in pregnant Wistar rats

Fully brominated diphenyl ether, decabromodiphenyl ether (DBDE), is one of the most widely used brominated flame retardants worldwide. Little data is available about the metabolic fate of DBDE in animal models and nothing at all about the extent of foetal exposure. In this work, pregnant Wistar rats were force-fed with 99.8% pure [14C]-DBDE over 96 h at a late stage of gestation (days 16 to 19). More than 19% of the administered dose was recovered in tissues and carcasses, demonstrating efficient absorption of DBDE despite its high molecular weight and low solubility. The highest concentrations of DBDE residues were found in endocrine glands (adrenals, ovaries) and in the liver, with lower values recorded for fat. In all tissue extracts, most of the radioactivity was associated with unchanged DBDE. The use of high-grade purity [14C]-DBDE allowed quantification of several metabolites present both in maternal tissues and in foetuses. These biotransformation products accounted for 9–27% of the extractable radioactivity in tissues and 14% of that in foetuses. Three nona-BDEs and one octa-BDE were identified by LC-APPI/MS. The unequivocal characterisation of a hydroxylated octa-BDE isolated from liver was confirmed by NMR. In rat, the main metabolic pathways of DBDE are debromination and oxidation. DBDE, and very likely most of its metabolites, are able to cross the placental barrier in rat. Metabolic profiles, obtained in vivo for the first time, demonstrated the presence of DBDE and major biotransformation products in endocrine glands as well as in foetuses. The biological activity of these metabolites still needs to be assessed in order to better understand the potential toxicity of DBDE.

Biotransformation of a Monoterpene Mixture by in vitro Cultures of Selected Conifer Species

Biotransformation of monoterpenes is a mild and promising method for the production of stereospecific and regiospecific organic compounds. It is advantageous, especially for the preparation of substances with complicated structures and for those that need to be classed as “natural products“. Our study has focused on the biotransformation of a monoterpenic mixture, turpentine, by Picea abies and Taxus baccata suspension cultures. We identified the biotransformation products and compared the metabolite compositions of the tissue cultures. The major biotransformation products of turpentine were trans-pinocarveol, trans-verbenol, verbenone, myrtenol, α-terpineol and trans-sobrerol, which correspond with the products of biotransformation of the individual monoterpenes, α-pinene and β-pinene, by P. abies suspension culture.

Enzymatic immobilization of 2,4,6-trinitrotoluene (TNT) biodegradation products onto model humic substances

The ability to enzymatically immobilize TNT biotransformation metabolites onto humic monomers was investigated using new laccase producing fungal isolates, namely: Trametes modesta BT2368, Trametes versicolor BT2570, Trametes hirsuta BT2566, T. hirsuta BT2133 and Sclerotium rolfsii CBS 350.80. Degradation of TNT by these organisms was confirmed using HPLC and it was shown to lead predominantly to biotransformation. Aminodinitrotoluenes (4-aminodinitrotoluene (4-AMDNT) and 2-aminodinitrotoluene (2-AMDNT)) were identified as the major biotransformation products. T. hirsuta BT2566 cultures supplemented with 220 μM TNT produced the highest amounts of 4-AMDNT and 2-AMDNT up to a concentration of 104 and 52 μM after 96 h of incubation, respectively. Incorporation of 200 mM of humic monomers especially ferulic acid, guaiacol and catechol during the biotransformation of TNT drastically reduced the accumulation of aminodinitrotoluenes to below 30%. In the presence of a purified laccase from T. hirsuta complete immobilization of 2-hydroxylaminodinitrotoluenes (2-HADNT) and 4-hydroxylaminodintrotoluenes (4-HADNT) by the different humic monomers was achieved. Ferulic acid was the most effective humic monomer in immobilizing TNT metabolites both in in vitro and culture experiments.

Biochemical changes in dehydrogenase, hydroxylase and tyrosinase of a permethrin-resistant strain of housefly larvae, Musca domestica L.

In the present study, a permethrin-resistant strain (ALHF) of housefly was used to understand some enzymic changes in normal biosynthetic pathways after insecticide selection. Aflatoxin B 1 (AFB 1) as a natural substrate was used to verify the changes on the level of cytochrome P450-dependent monooxygenases and oxido-reductase activities in the ALHF strain compared to an insecticide-susceptible strain, aabys. ALHF yielded three major biotransformation products: aflatoxin B 2a (AFB 2a), aflatoxin M 1 (AFM 1), and aflatoxicol (AFL) by larvae. These principal products were also found in aabys. AFL production rate of ALHF larvae was 5-fold lower than that of aabys. Differences between ALHF larvae and aabys in AFM 1 production were found. ALHF did not differ significantly from aabys in AFB 2a production. The levels of 17α- and β-hydroxysteroid dehydrogenase (17α- and β-HSD) were also determined to elucidate which type of dehydrogenase activities could be changed. The cytosolic fraction of ALHF larvae yielded about 2-fold higher 17α-estradiol than that of aabys larvae. In contrast, the microsomal fraction of ALHF larvae produced about 2-fold lower amount of 17α-estradiol than that of aabys larvae. The production rate of microsomal fraction of 17β-estradiol ALHF larvae yielded 3-fold lower than that of aabys larvae. Inhibition studies on 17α-HSD and 17β-HSD activities by pyrethroid insecticides showed that there was no inhibition by pyrethroids on the enzyme activity. Therefore, there seems to be no changes on the enzyme structures. Changes on enzyme expression may occur in ALHF larvae in relation to 17α- or β-HSD. To assess biochemical changes of the cuticle formation phenylalanine 4-hydroxylase and tyrosinase activities were determined. The production rate of tyrosine from phenylalanine in ALHF was about 2-fold higher for larvae than that in aabys. l-(dihydroxylphenyl)alanine (DOPA) content was determined in larvae and ALHF possessed 1.6-fold larger amounts of DOPA than aabys. Tyrosinase activity of ALHF larval preparations showed 1.6-fold higher than aabys. In summary, many enzymic changes were found in ALHF strain compared to aabys strain and these changes may be resulted from the permethrin selection.