Biotransformation of albendazole by Cunninghamella blakesleeana: effect of carbon and nitrogen source

A filamentous fungus Cunninghamella blakesleeana was screened for its ability to biotransform the anthelmintic drug albendazole. The fungus produced three metabolites in the presence of the carbon and nitrogen sources studied. The transformation was identified by HPLC and the structures of the transformation products were assigned by LC-MS-MS analysis and on the basis of previous reports. The results indicated that the fungus metabolized albendazole into albendazole sulfoxide (M1), albendazole sulfone (M2) and an N-methyl metabolite of albendazole sulfoxide (M3). The effect of carbon and nitrogen source on the biotransformation of albendazole was significant. Among the carbon and nitrogen sources studied, fructose and urea respectively produced maximum extent of biotransformation in terms of substrate depletion. Among the carbon sources studied, maltose produced a higher percentage of M1 whereas M2 and M3 were produced to their maximum extent in presence of d-fructose in terms of metabolite per unit quantity of biomass. In the case of nitrogen sources, ammonium acetate, calcium nitrate and barium nitrate produced maximum percentage of M1, M2 and M3 respectively. The results reveal that the carbon and nitrogen source significantly influence the microbial transformation both qualitatively and quantitatively.     

Simultaneous liquid chromatography-tandem mass spectrometric determination of albendazole sulfoxide and albendazole sulfone in plasma

This paper describes a simple, fast, sensitive and reliable method for the simultaneous determination of albendazole sulfoxide (ASOX) and albendazole sulfone (ASON), the two most important metabolites of the drug albendazole (ABZ), in plasma samples using liquid chromatography and tandem mass spectrometry. After liquid-liquid extraction with dichloromethane, the two albendazole metabolites and the internal standard phenacetin were resolved in a CN column using the mobile phase methanol-water (4:6, v/v) acidified with 1% acetic acid. Detection by electrospray mass spectrometry was carried out in the positive ion mode. The method was linear up to 2500 and 250 ng/ml for ASOX and ASON, respectively, with mean recoveries of more than 85%. The precision and accuracy data, based on within- and between-day variations over 5 days, were lower than 15%. The quantitation limits of 0.5 and 5.0 ng/ml for ASON and ASOX are low enough for the method to be suitable for pharmacokinetic studies. Pharmacokinetic data obtained with the proposed method following oral administration of ABZ to a patient with neurocysticercosis are also reported.     

Simultaneous determination of albendazole sulfoxide enantiomers and albendazole sulfone in plasma

A high-performance liquid chromatographic method has been developed for the simultaneous determination of albendazole sulfoxide (ABZSO) enantiomers and albendazole sulfone (ABZSO₂) in human plasma. The resolution of ABZSO enantiomers and ABZSO₂ was obtained on a Chiralpak^® AD column using hexane–isopropanol–ethanol (81:14.25:4.75, v/v/v) as the mobile phase. The drugs were detected by fluorescence (λ_exc=280 nm, λ_em=320 nm). The drugs were extracted from 500 μl plasma with ethyl acetate, and after solvent evaporation, the residues were dissolved in the mobile phase and chromatographed. The method was precise and accurate for the three compounds, as judged by the coefficients of variation and relative errors observed. Linear standard curves were obtained in the concentration range of 5–2500 ng/ml for ABZSO enantiomers and 1–500 ng/ml for ABZSO₂. A typical plasma concentration–time profile is presented for one patient under treatment for neurocysticercosis.     

Trace analysis of albendazole and its sulphoxide and sulphone metabolites in milk by liquid chromatography

Analytical methodology for determination of albendazole and its sulphoxide and sulphone metabolites in milk at levels down to 2–5 ng/ml has been developed. Extraction was carried out with ethyl acetate under alkaline conditions, and extracts were analyzed on a silica-based C₁₈ column in the presence of positively-charged pairing ions. Accuracy data showed overall recoveries ranged from 78.4% to 100%, whereas precision data, based on within- and between-day variation, suggested overall precision values better than 4.9%. The method was successfully applied to determine residues in milk of a dairy cow orally given albendazole.     

In vitro evaluation of the effects of cysticidal drugs in the Taenia crassiceps cysticerci ORF strain using the fluorescent CellTracker CMFDA

Using a murine model of cysticercosis caused by the Taenia crassiceps ORF strain, we developed a fluorescent quantitative evaluation of the action of two well known anti-helminthic drugs: albendazole sulfoxide and praziquantel. The fluorescence emitted by a biotransformed CellTracker Probe known as CellTracker Green CMFDA in the vesicular fluids of cysticerci was estimated, and the results were compared with macroscopic observations of the parasites. The pharmacological EC₅₀ value of each drug and changes in the level of biotransformation of the fluorescent tracker caused by the drugs could be easily calculated. These drug-induced changes in biotransformation could be related to changes in the GSH/GSSG ratio of parasites. Both the cysticercosis murine model and the CMFDA biotransformation assay could be used as an in vitro screening method to evaluate potential or well known cysticidal drugs.     

In vivo evaluation of the efficacy of albendazole sulfoxide and albendazole sulfoxide loaded solid lipid nanoparticles against hydatid cyst

Cystic echinococcosis (CE) is caused by the larval stage of Echinococcus granulosus, which in this disease the metacestode develop in visceral organs especially liver and lungs. The disease is present worldwide and affects humans as well as herbivores including cattle, sheep, camels, horses and others. Benzimidazole carbamate derivatives, such as mebendazole and albendazole, are currently used for chemotherapeutic treatment of CE in inoperable patients and have to be applied in high doses for extended periods of time, and therefore adverse side effects are frequently observed. This study was designed to evaluate and compare the in vivo effects of 0.5 mg/kg, BID, albendazole sulfoxide (ricobendazole) and two different therapeutic regimens of 0.5 mg/kg BID and 2 mg/kg every 48 h of albendazole sulfoxide loaded solid lipid nanoparticles. Albendazole sulfoxide loaded solid lipid nanoparticles was prepared by solvent diffusion–evaporation method. Fifty Balb/c mice were infected by intraperitoneal injection of protoscoleces and 8 months post infection, the infected mice were treated for 15 days with the above mentioned regimens. They were then euthanized and the size and weight of the cysts as well as their ultrastructural changes were investigated. Although the cysts showed reduced size and weight in the treated animals but these reductions were not statistically significant. The cysts in the animals which received albendazole sulfoxide loaded SLN every 48 h showed more ultrastructural modification. However, these ultrastructural changes should be supported by further biochemical and molecular studies before introducing it as an efficient therapeutic regimen for treatment of human and animal hydatid disease.     

Microbial metabolism and detoxification of 7,12-dimethylbenz[a]anthracene

Summary Six strains of fungi grown on Sabouraud dextrose broth in the presence of 7,12-dimethylbenz[a]anthracene (DMBA) were surveyed for their ability to metabolize DMBA. Experiments with [¹⁴C]DMBA indicated that the extent of formation of organic-soluble metabolites ranged from 6 to 28% after 5 days of incubation, depending on the organism tested. The yields of water-soluble metabolites also varied, and ranged from 1 to 33% after 5 days.Cunninghamella elegans ATCC 36112 andSyncephalastrum racemosum UT-70 exhibited the highest DMBA-metabolizing activity among the organisms surveyed.S. racemosum metabolized DMBA primarily to 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA)_ and 7,12-dihydroxymethylbenz[a]anthracene (7,12-diOHMBA). Minor metabolites included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols, and glucuronide and sulfate conjugates of phenolic derivatives of DMBA. In contrast, the major DMBA metabolites produced byC. elegans were water-soluble. The predominant organic-soluble metabolites produced byC. elegans included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols. DMBA-trans-3,4-dihydrodiol was also detected. Circular dichroism spectral analysis revealed that the major enantiomer of the 7-OHM-12-MBA-trans-8,9-dihydrodiol formed by each organism has anS,S absolute configuration, while the major enantiomers of the 5,6-, 10,11- and 3,4-dihydrodiols had anR,R configuration. The mutagenic activity of extracts fromS. racemosum exposed to DMBA were determined inSalmonella typhimurium TA98. The mutagenicity of DMBA decreased by 36% over a period of 5 days as 33% of the compound was metabolized. Comparison of these results with previously reported results in mammalian systems suggests that there are similarities and differences between the fungal and mammalian oxidation of DMBA and that the overall balance of fungal metabolism is towards a detoxification rather than a bioactivation pathway.     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes

To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was tested with six A.␣tumefaciens strains, one plasmid (pLC101) and the doubleCaMV35s (dCaMV35s) promoter. The CaMV35s promoter produced more GUS expression than the CAB promoter. A. tumefaciens strains EHA105 and LBA4404 harboring the same plasmid (pBIN19) differed in their effects on GUS expression suggesting an interaction between A. tumefaciens strain and plasmid. A combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter produced the highest rates of transformation in peach epicotyl internodes (56.8%), cotyledons (52.7%), leaves (20%), and embryonic axes (46.7%) as evaluated by the percentage of explants expressing GUS 14 days after co-cultivation. GFP expression under the control of the dCaMV35s promoter was highest for internode explants but only reached levels of 18–19%. When GFP-containing plasmid pCL101 was combined with each of five A. tumefaciens strains the highest levels of transformation were 20–21% (internode and cotyledons, respectively). When nine peach genotypes were co-cultivated with A. tumefaciens strain EHA105 and GFP-containing plasmid pCL101 the highest levels of transformation were 26–28% (cotyledons and internodes, respectively). While GFP represents a potentially useful transformation marker that allows the non-destructive evaluation of transformation, rates of GFP transformation under the conditions of this study were low. It will be necessary to optimize expression of this marker gene in peach.     

Anaerobic induced ethanol synthesis in the stems of greenhouse-grown conifer seedlings

Ethanol synthesis was induced in stem segments from greenhouse-grown conifer seedlings by placing them in a N₂ atmosphere at 30 °C for 24 h. Stems from ponderosa pine,Pinus ponderosa Dougl. ex Laws., sugar pine,Pinus lambertiana Dougl., Pacific silver fir,Abies amabalis Dougl. ex Forbes, and lodgepole pine,Pinus contorta Dougl. ex Loud, produced the highest quantities of ethanol. This group also had the smallest and slowest growing stems. Within each of these species the amount of ethanol produced was inversely related to the stem volume. Stems from western hemlock,Tsuga heterophylla (Raf.) Sarg., grand fir, Abies grandis Dougl. ex Forbes, Douglas-fir,Pseudotsuga menziesii (Mirb.) Franco, and western redcedar,Thuja plicata Donn ex D. Don, all produced equivalent but low ethanol concentrations. These species had the largest and fastest growing stems. In this group only grand fir exhibited an inverse relationship between ethanol concentrations and stem volume. The relative amounts of ethanol synthesized by stems from Douglas-fir, western hemlock and western redcedar seedlings were not the same as subsequently observed in logs from mature trees of the same species under field conditions. Differences in the anaerobic environments for the two stem types could have affected the quantities of ethanol produced. The observed high amounts of ethanol produced by the stems from pine species were discussed in terms of their ability to handle periods of anaerobic stress or hypoxia.     

Isolation and identification of Paenibacillus sp. FM-6, involved in the biotransformation of albendazole

A strain, designated as FM-6, was isolated from fish. Based on the results of phenotypic, physiological characteristics, genotypic and phylogenetic analysis, strain FM-6 was finally identified as Paenibacillus sp. When albendazole was provided as the sole carbon source, strain FM-6 could grow and transform albendazole. About 82.7 % albendazole (50 mg/L) was transformed by strain FM-6 after 5 days incubation at 30 °C, 160 rpm. With HPLC–MS method, the transforming product of albendazole was researched. Based on the molecular weight and the retention time, product was identified as albendazole sulfoxide and the transforming pathway of albendazole by strain FM-6 was proposed finally. The optimum temperature and pH for the bacterium growth and albendazole transformation by strain FM-6 were both 30 °C and 7.0. Moreover, the optimum concentration of albendazole for the bacterium growth was 50 mg/L. Coupled with practical production, 50 mg/L was the optimum concentration of albendazole transformation for strain FM-6. This study highlights an important potential use of strain FM-6 for producing albendazole sulfoxide.     

Determination of Albendazole and Metabolites in Silkworm Bombyx mori Hemolymph by Ultrafast Liquid Chromatography Tandem Triple Quadrupole Mass Spectrometry

Albendazole is a broad-spectrum parasiticide with high effectiveness and low host toxicity. No method is currently available for measuring albendazole and its metabolites in silkworm hemolymph. This study describes a rapid, selective, sensitive, synchronous and reliable detection method for albendazole and its metabolites in silkworm hemolymph using ultrafast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-MS/MS). The method is liquid-liquid extraction followed by UFLC separation and quantification in an MS/MS system with positive electrospray ionization in multiple reaction monitoring mode. Precursor-to-product ion transitions were monitored at 266.100 to 234.100 for albendazole (ABZ), 282.200 to 208.100 for albendazole sulfoxide (ABZSO), 298.200 to 159.100 for albendazole sulfone (ABZSO₂) and 240.200 to 133.100 for albendazole amino sulfone (ABZSO₂-NH₂). Calibration curves had good linearities with R² of 0.9905–0.9972. Limits of quantitation (LOQs) were 1.32 ng/mL for ABZ, 16.67 ng/mL for ABZSO, 0.76 ng/mL for ABZSO₂ and 5.94 ng/mL for ABZSO₂-NH₂. Recoveries were 93.12%–103.83% for ABZ, 66.51%–108.51% for ABZSO, 96.85%–105.6% for ABZSO₂ and 96.46%–106.14% for ABZSO₂-NH₂, (RSDs <8%). Accuracy, precision and stability tests showed acceptable variation in quality control (QC) samples. This analytical method successfully determined albendazole and its metabolites in silkworm hemolymph in a pharmacokinetic study. The results of single-dose treatment suggested that the concentrations of ABZ, ABZSO and ABZSO₂ increased and then fell, while ABZSO₂-NH₂ level was low without obvious change. Different trends were observed for multi-dose treatment, with concentrations of ABZSO and ABZSO₂ rising over time.     

Zataria multiflora would attenuate the hepatotoxicity of long-term albendazole treatment in mice with cystic echinococcosis

Hepatic injury is the major limitation of long-term albendazole administration in patients with cystic echinococcosis (CE), which could give rise to cessation of treatment. The objective of the present study was to evaluate the protective effects of Zataria multiflora aromatic water (AW) against the hepatic injury induced by long-term albendazole treatment in mice with CE. Fifty healthy BALB/c female mice were infected intraperitoneally by injection of 1500 protoscoleces per animal. Five months after infection, the infected animals were divided into five treatment groups including Z. multiflora (40 ml/l in drinking water for 90 days), albendazole (200 mg/kg/day for 90 days), Z. multiflora + albendazole 200 (40 ml/l Z. multiflora and 200 mg/kg/day albendazole for 90 days), Z. multiflora + albendazole100 (40 ml/l Z. multiflora and 100 mg/kg/day albendazole for 90 days), and untreated (control) group. At the end of the treatment period, anesthesia was performed and blood samples were collected directly from the heart prior to euthanasia. Liver variables and oxidative stress markers were measured in the blood serum samples. A decrease in serum liver enzyme activity in the both Z. multiflora + albendazole groups was observed when compared to control, Z. multiflora and albendazole groups; however, the results for Z. multiflora + albendazole 100 were significant (p < 0.007) and superior compared to those for Z. multiflora + albendazole 200. No significant differences for oxidative stress markers were observed between the different groups. The results of the present study revealed that a combined therapy with Z. multiflora AW and albendazole is effective against hepatic injury induced by CE and/or long term albendazole administration in mice with cystic echinococcosis.     

The production of aflatoxin M on various substrates

Summary The ability of 44 strains ofA. flavus and 6 strains ofA. parasiticus to produce aflatoxin M on various substrates was examined. It was found that these strains produced aflatoxin M only with larger quantities of aflatoxin B. The presence of several other minor metabolites in culture extracts is described. The highest yield of aflatoxin M was produced by a strain ofA. flavus grown on maize meal.     

Cytotoxicity of Fusarium species mycotoxins and culture filtrates of Fusarium species isolated from the medicinal plant Tribulus terrestris to mammalian cells

Ayurvedic medicine, which uses decoctions made of medicinal plants, is used to cure diseases in many Asian countries including Sri Lanka. Although proper storage facilities for medicinal plants are unavailable in Sri Lanka, neither the potential for growth of toxigenic fungi nor their ability to produce mycotoxins in stored medicinal plants has been investigated. We isolated three Fusarium species, F. culmorum, F. acuminatum and F. graminearum from the medicinal plant Tribulus terrestris. Culture extracts of the 3 Fusarium spp. were cytotoxic to mammalian cell lines BHK-21 and HEP-2. Three toxic metabolites produced by Fusarium spp; T-2 toxin, zearalenone, and diacetoxyscirpenol were also cytotoxic to the same mammalian cell lines. The 3 Fusarium spp. grown on rice media produced zearalenone. Plant material destined for medicinal use should be stored under suitable conditions to prevent growth of naturally occurring toxigenic fungi prior to its use.     

Mould growth on building materials under low water activities. Influence of humidity and temperature on fungal growth and secondary metabolism

The influence of relative humidity (RH) and temperature on growth and metabolism of eight microfungi on 21 different types of building material was investigated. The fungi were applied as a dry mixture to the materials, which were incubated at 5°C, 10°C, 20°C and 25°C at three humidity levels in the range 69–95% RH over 4–7 months. The lower limit for fungal growth on wood, wood composites and starch-containing materials was 78% RH at 20–25°C and increased to 90% RH at 5°C. An RH of 86% was necessary for growth on gypsum board. Ceramic materials supported growth at RH >90%, although 95% RH was needed to yield chemically detectable quantities of biomass. Almost exclusively only Penicillium, Aspergillus and Eurotium (contaminant) species grew on the materials. Production of secondary metabolites and mycotoxins decreased with humidity and the quantities of metabolites were insignificant compared with those produced at high RH (RH >95%), except in the case of Eurotium.     

Influence of cadmium on certain biological activities in a cadmium-tolerant fungi

Aspergillus carbonarius and a strain ofPenicillium are able to grow on Harrold's agar media amended with different concentrations of cadmium chloride up to 2.5% (w/v). Considerable quantities of cadmium were absorbed by both fungi.A. carbonarius absorbed more cadmium than thePenicillium sp. did, under the same culturing conditions. In the presence of cadmium, the determined cellular contents of proteins, lipids, and carbohydrates were extraordinary high, whereas the activities of certain enzymes, lipases, amylases, and proteases were inhibited. The fungal rate of growth and sporulations were mostly suppressed. Conidiations were inhibited at lowest concentrations. At 1% Cd Cl₂,A. carbonarius produced malformed conidiophores, whereas thePenicillium sp. was less affected. At higher concentrations conidiophores production were entirely suppressed and several hyphal swellings were produced.     

Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on GFP fluorescence with a very high accuracy. The method requires no selection agents like antibiotics or herbicides and enables a reduction of labour and time necessary for tissue culture. Moreover, individual transformants can be easily excised from the host tissue and cultured separately. All of the 12 used carrot cultivars produced transformed adventitious roots and the frequency of discs producing GFP expressing adventitious roots varied from 13 to 85%. The highest transformation rate was found for A4T and LBA1334 strains possessing chromosomal background of A. tumefaciens C58. The results encourage that visual selection of transformed, fluorescing adventitious roots can be highly effective and applied routinely for the production of carrot transgenic plants.     

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology.     

Production of human metabolites of the anti-cancer drug flutamide via biotransformation in <Emphasis Type="Italic">Cunninghamella</Emphasis> species

Fungi belonging to the genus Cunninghamella have enzymes similar to those employed by mammals for the detoxification of xenobiotics, thus they are useful as models of mammalian drug metabolism, and as a source for drug metabolites. We report the transformation of the anti-cancer drug flutamide in Cunninghamella sp. The most predominant phase I metabolites present in the plasma of humans, 2-hydroxyflutamide and 4-nitro-3-(trifluoromethyl)aniline, were also produced in Cunninghamella cultures. Other phase I and phase II metabolites were also detected using a combination of HPLC, GC–MS and ¹⁹F-NMR.