The expression of cytochrome P-450 and cytochrome P-450 reductase (CPR) genes in the conterminous biotransformation of corticosteroids and PAHs was studied in Cunninghamella elegans 1785/21Gp. We had previously used this strain as a microbial eucaryotic model for studying the relationship between mammalian steroid hydroxylation and the metabolization of PAHs. We reported that cytochrome P-450 reductase is involved in the biotransformaton of cortexolone and phenanthrene. RT-PCR and Northern blotting analyses indicated that the cytochrome P-450 and CPR genes appear to be inducible by both steroids and PAHs. The expression of the cytochrome P-450 gene was increased ninefold and the expression of the CPR gene increased 6.4-fold in cultures with cortexolone and/or phenanthrene in comparison with controls. We conclude that the increase in cytochrome P-450 gene expression was accompanied by an increase in cytochrome P-450 enzymatic activity levels.
Filamentous fungi are able to oxidize different compounds using mechanisms which are similar to those observed in the mammalian system (Casillas, 1996). Xenobiotic biotransformation by microbial cells can be used to predict mammalian xenobiotic metabolism (Srisilam & Veeresham, 2003). The zygomycete fungus Cunninghamella sp. has been profitably applied in research as a microbial model for the mammalian metabolism of xenobiotics, due to its ability to oxidize different compounds using cytochrome P-450 enzymes (Cerniglia, 1992). We have previously documented that Cunninghamella elegans IM 1785/21Gp has an ability to efficiently biotransform phenanthrene and cortexolone (Lisowska & Długoński, 1999, 2003). Phenanthrene is a polycyclic aromatic hydrocarbon (PAH) which is often found in polluted environments and is useful as a PAH model for the study of mammalian (Grimmer, 1991) and fungal (Bezalel, 1997; Boehmer, 1998) metabolism, because it is thought to be noncarcinogenic and nonmutagenic, as well as having a fused-ring structure similar to higher molecular weight carcinogenic PAHs (Samanta, 2002). Cortexolone is one of the most important substrates for the production of bulk quantities of hydrocortisone and other mammalian steroid hormones in industry (Sedlaczek, 1988).
We have previously reported that cytochrome P-450 is involved in the biotransformation of both types of lipophilic substrates (phenanthrene and cortexolone) by C. elegans (Lisowska & Długoński, 2003). Cytochrome P-450 is part of the mono-oxygenase enzyme system. These systems are multicomponent, consisting of both P-450 and electron donating parts, needed for oxygen insertion in the substrate molecule (Makovec & Breskwar, 2000). A common electron donor to the multiple P-450 mono-oxygenases is cytochrome P-450 reductase (CPR) (Yadav & Loper, 2000a). This enzyme contains one molecule of both FAD and FMN. The electron transfer proceeds from NADPH to FAD, to FMN in the CPR, to the P-450 heme. The CPR gene has been isolated from selected animal species, insects, plants and yeasts. However, amongst filamentous fungi the gene has only been reported for a few species (Yadav & Loper, 2000b). In this paper we present data on the involvement of cytochrome P-450 reductase in the coincident biotransformation of cortexolone and phenanthrene by C. elegans. In addition, we have extended our previous studies (Lisowska & Długoński, 2003; Lisowska, 2006) on cytochrome P-450 activity at the enzymatic level.
Materials and methods
Phenanthrene and cortexolone (17α,21-dihydroxy-4-pregnene-3,20-dione) were obtained from Sigma-Aldrich Inc. (St Louis, MO). Solvents were purchased from J.T. Baker, Serva and POCH (Poland). All the chemicals and solvents were high purity grade reagents.
Stock solutions of phenanthrene were prepared in dimethylformamide (DMF) at a concentration of 12.5mgmL−1. Cortexolone was dissolved in ethanol to give a final concentration of 1–2% (v/v) in the culture.
Cunninghamella elegans IM 1785/21Gp able to carry out cortexolone hydroxylation in positions 11α-, and 11β-, yielding epihydrocortisone and hydrocortisone, respectively (Długoński, 1991; Długoński, 1997) and PAHs degradation (Lisowska & Długoński, 1999) was used. Trichoderma viride 1131 CBS 354–33 was applied as a source of lytic enzymes, which digest C. elegans cell wall (Długoński, 1991; Wilmańska, 1992).
Corticosteroid and phenanthrene metabolization
Preculturing, steroid and phenanthrene biotransformation were performed at 28°C. Ten-day cultures on Sabouraud agar slants were used to inoculate 20mL of Sabouraud liquid medium (10g of neopeptone [Difco Laboratories, Detroit, MI], and 40g glucose L−1 deionized water). The cultivation was carried out in 100mL Erlenmayer flasks for 24h. The preculture (2mL) was transferred to fresh medium (at a 1:9 ratio) and incubated for the next 24h. The homogenous precultures of the fungi thus obtained (2mL) were reintroduced to the fresh medium (at a 1:9 ratio), and a steroid substrate (cortexolone) dissolved in ethanol (95% v/v) (0.5gL−1) or/and phenanthrene dissolved in DMF (0.25gL−1) were added, at different times after inoculation, as indicated in the text. The flasks were incubated for 7 days.
Release of protoplasts
The samples from the phenanthrene and cortexolone cultivations (as indicated in the text) were filtered and washed twice with distilled water, then the mycelium (100mg) was suspended in 5mL citrate-phosphate buffer, pH 4.2, containing 0.8M MgSO4. Lytic enzymes, obtained as described earlier (Lisowska & Długoński, 2003) (2.5mglyophilizedpreparationmL−1) were added and the digestion was terminated when no further increase in the number of protoplasts was observed (12–20h). The digestion mixture was filtered through a nylon net, transferred into 0.6M KCl and counted under a microscope (Długoński, 1984).
Preparation of total RNA
Total cellular RNA was extracted from the protoplasts using the TRIzol reagent method (Invitrogen, Carlsbad, CA) with a single-step purification protocol (Chomczyñski & Sacchi, 1987). RNA pellets were dissolved in DEPC water and their concentrations and purity were determined by spectrophotometer (DU-640B; Beckmann Instruments, Palo Alto) with readings at 260 and 280nm. The 260/280 ratios were always greater than 1.8. The integrity of the RNA samples was checked on a 1% agarose gel. The RNA was stored at −80°C.
Reverse transcriptase and PCR (RT-PCR)
cDNA was synthesized from 2.5μg of isolated total RNA using AMV Reverse Transcriptase (Promega Corpet al, Madison, WI) and random hexamers (500μgmL−1) according to the manufacturer's protocol. The same amounts of specific mRNA were quantified by PCR obtained cDNA. One-tenth of each RT reaction was amplified in a 25μL PCR mix containing 0.3μM of following primers: 5′-GGT ATG AAC TTT AGT TTA TTA GAA CAA-3′, 5′-CGG ATT TTC AAA TCA ATT GGT TTA GGT-3′ specific for cytochrome P-450, and 5′-GTA CAA CAT CGT TTA GAA GAA AAT GGT-3′, 5′-CAT CTT CTT GAT AAC GAC CAG TAT TAC-3′ specific for CPR and 5′-GAACTGTTCCCCGACCCCTACGG-3′, 5′-GAGCGTCACGAAGCCACGCCAAC-3′ specific for GADPH. The samples were incubated for 5min at 94°C for the initial denaturation and polymerase activation, followed by 35 PCR cycles of 94°C for 30s 56°C for 30s and 72°C for 30s. To compensate for variations in the amounts of input RNA, the efficiency of the reverse transcription GADPH mRNA was quantified and the results were normalized to the values mentioned above. The final products were separated by electrophoresis in 7% polyacrylamide gels in Tris-acetate-EDTA buffer. Bands were stained with ethidium bromide and visualized by UV light; the results were recorded photographically and analyzed densitometrically using an ImageMaster VDS system (Pharmacia Biotech). The analyses were performed in duplicate. The PCR products were sequenced in both directions using an automatic DNA sequencer system (Institute of Biochemistry and Biophysics, Poland), and the sequences were analyzed using the programs clustalw (http://www.ebi.ac.uk/clustalw/) and basicblast (http://www.ncbi.nih.gov/BLAST).
Northern blotting analysis
Ten micrograms of total RNA were size-fractionated by 1% agarose gel electrophoresis with 2M formaldehyde and transferred onto a positively charged nylon membrane Magna Graph (MSI, Westboro, MA). Cytochrome P-450, CPR and GADPH mRNAs were detected by hybridization with labelled 32P α-ATP PCR products specific for each gene. PCR products were labelled with 32P α-ATP (1 × 108dpmmL−1 hybridization buffer) using a random-priming method with random hexamers and Klenow fragment DNA polymerase I using a Prime-a-Gene labelling kit (Promega). Prehybridization and hybridization were carried out in hybridization buffer (5 × SSC, 5 × Denhardt's solution, 10% dextran sulfate, 1% SDS, 20μgmL−1 salmon sperm DNA in 50% formamide) overnight at 55°C. After hybridization the membranes were washed in 2 × SSC at room temperature for 30min, and then washed under highly stringent conditions (0.1 SSC, 1% SDS) at 65°C for 15min. Autoradiography was performed at −80°C overnight using Kodak XAR film. The membranes were then stripped and re-probed for the next hybridization. The intensity of the isotope signal was quantified using an ImageMaster VDS system (Pharmacia Biotech).
RT-PCR of the cytochrome P-450 and CPR genes
All the cytochrome P-450 genes contain a highly conserved heme-binding region (Gonzales, 1989; Wang, 2000). There are also highly conserved domains in the CPR genes which are characteristic of the different CPRs (Yadav & Loper, 2000a). Based on these regions, the expression of cytochrome P-450 and CPR genes was determined by RT-PCR. A genomic segment of the cytochrome P-450 and CPR genes was isolated from C. elegans by RT-PCR amplification of the total RNA using the primers described in the Materials and methods section.
Our previous results demonstrated an increase in cytochrome P-450 activity from the culture with cortexolone and phenanthrene, and much a greater level of cytochrome P-450 in the presence of both substrates. On the basis of this finding the following experiment was performed. The total RNA for reverse transcription was isolated from protoplasts obtained from the C. elegans culture with cortexolone, phenanthrene and phenanthrene in the presence of cortexolone (which was added to the culture 12h after inoculation with PAH) and cortexolone in the presence of phenanthrene (added to the culture 12h after inoculation with steroid). A culture of C. elegans without any additions served as a control. To characterise the expression of both genes in C. elegans we analysed the levels of cytochrome P-450 and CPR mRNA using a semi-quantitative RT-PCR method based on the use of different cultures (mentioned above) during the incubation period.
PCR products of the expected size (148bp) were detected on a polyacrylamide gel after RT-PCR with total RNA extracted from the control and cultures with cortexolone or/and phenanthrene, indicating the presence of cytochrome P-450 mRNA. (Fig. 1a). Direct sequencing of the amplicons yielded a distinct sequence of 148bp in all cases. Similarity screening demonstrated that this sequence was related to the cytochrome P-450 C. elegans gene (GenBank accession no. AF249299). On the basis of the cDNA amplification fragments we can state that expression of the cytochrome P-450 gene in control cultures of C. elegans (without corticosteroids and PAH addition) was very low. A clear, but weak signal was evident in the control samples throughout the 96-h incubation. The addition of steroid or PAH substrate gave rise to 6.3-fold and 4.3-fold increases, respectively (in 24h of incubation) in the expression of cytochrome P-450 genes. Moreover, the C. elegans cultivated in the presence of both substrates showed the highest cytochrome P-450 gene expression. This dependence was most strongly observed in 24 of the C. elegans cultures with cortexolone and phenanthrene. In this case, the signal was 9-fold greater than the control (without any substrate addition).
The C. elegans control culture and the culture cultivated with cortexolone or/and phenanthrene throughout the incubation gave a positive signal for CPR mRNA in RT-PCR analysis, as evidenced by an amplified fragment of the expected 210bp size on the polyacrylamide gel (Fig. 1b). Sequencing in both directions of the PCR products obtained from control samples and samples with additional cortexolone and phenanthrene yielded a sequence of 210bp. The alignment analysis of these sequences showed a similarity to the CPR C. elegans gene (GenBank accession no. AF195659).
CPR expression was clearly higher in the culture with additional steroid or phenanthrene, compared to the control (without cortcosteroids or PAH). The highest levels of CPR mRNA were observed for both cortexolone and phenanthrene addition in the 24-h incubation. In this case the transcripted amount was approximately 6.4-fold higher than that measured from controls. These results demonstrate the involvement of CPR in both cortexolone and phenanthrene metabolism.
The expression of GADPH dehydrogenase genes (the model constitutive enzyme) remained the same throughout the whole incubation (Fig. 1c).
Northern-blotting analysis of cytochrome P-450 and CPR genes
The results obtained from the RT-PCR were further substantiated by a Northern-blotting analysis of cytochrome P-450 and CPR gene expression in C. elegans.
A high level of cytochrome P-450 mRNA was formed in the C. elegans culture with additional cortexolone or phenanthrene in 24h of the incubation (Fig. 2). The signal was about ninefold greater than that of the control. The highest levels were observed in cultures cultivated with both phenanthrene and cortexolone, about 13-fold higher than in controls. This dependence was also observed for CPR gene expression. The addition of both cortexolone and phenanthrene revealed an approximately 10-fold increase in mRNA level compared with control.
The results of the Northern blotting analysis are comparable to those obtained from the RT-PCR method. Both methods showed that the CPR as well as the cytochrome P-450 genes appear to be inducible by steroids and PAHs.
Cytochromes P-450s are biotransformation phase I enzymes that play important roles in the metabolism of xenobiotics including polyaromatic hydrocarbons (Kim, 2004) and steroids (Breskvar, 1995; Makovec & Breskwar, 2000). Because it is known that the metabolism of different xenobiotics by C. elegans is similar to that in mammals (Casillas, 1996; Yadav & Loper, 2000a), in this work the involvement of cytochrome P-450 reductase in the coincident biotransformation of corticosteroids and PAHs was studied in C. elegans.
A mammalian microsomal system usually contains both a mono-oxygenase and a reductase component (Yadav & Loper, 2000a). However, the presence of a two-component microsomal P-450 system in Eukaryotes is not a general rule. Breskvar (1987) reported that the 11α hydroxylation of progesterone by Rhizopus nigricans was mediated by three compounds: NADPH-dependent rhizoporedoxine reductase, rhizoporedoxine and cytochrome P-450. Guengerich (1992) also described a three-component system in the 11-β hydroxylation of deoxycorticosterone by adrenal cortex cells. In contrast to the two-component cytochrome P-450 enzyme system, which is often found in Eukaryotes, the cytochrome P-450 enzyme system in Fusarium oxysporum was shown to be a single, membrane-bound, fusion protein (Nakayama, 1996). We reported that in the biotransformation of both cortexolone and phenanthrene by C. elegans IM 1785/21Gp the cytochrome P-450 system consists of two components: cytochrome P-450 reductase and cytochrome P-450.
Expression of the cytochrome P-450 system can be induced, for example, by n-alkanes (Ohkuma, 1995), or benzoate (van den Brink, 1995). Yadav and Loper (2000a) showed that the CPR gene was transcriptionally expressed in C. elegans and appeared to be inducible by an alkane substrate, n-tetradecane. However, the substrate induction of specific P-450 enzyme systems (cytochrome P-450 and CPR), in filamentous fungi may not be a general feature (van den Brink, 1998). The treatment of mycelium from A. ochraceus and A. parasiticus with the general P-450 inducer phenobarbital resulted in a three- to fivefold increase in CPR and benzo(a)pyrene hydroxylase activities. However, phenobarbital or benzo(a)pyrene treatment did not affect CPR activity in Aspergillus fumigatus or cpr mRNA levels in Aspergillus niger (van den Brink, 1998). In the present study, RT-PCR and Northern blotting analysis indicated that the cytochrome P-450 and CPR genes appear to be inducible by both steroids and PAHs. In this study we have demonstrated an increase in cytochrome P-450 and CPR mRNA levels in the presence of cortexolone and phenanthrene. The expression of the cytochrome P-450 gene was increased ninefold and the expression of the CPR gene increased 6.4-fold in cultures having additional cortexolone and/or phenanthrene, in comparison with the controls, measured by a semi-quantitative RT-PCR. The present investigation extends our previous findings from P-450 activity on the enzymatic level in C. elegans IM 1785/21Gp (Lisowska & Długoński, 2003). Thus, we can state that the increase in cytochrome P-450 gene expression is accompanied with an increase in cytochrome P-450 enzymatic activity. However, such a dependence is not a general rule. In A. niger, the increase in cytochrome P-450 reductase, cprA and cytochrome P-450 genes, and bphA mRNA level were not correlated with an increase in CPR and BPH activity (van Gorcom, 1990; van den Brink, 1995, 1996).
Until now there have been no data on the involvement of the CPR enzyme in the simultaneous bioconversion of cortexolone and phenanthrene. Our study makes an important step towards an understanding of the metabolic controls that regulate the cytochrome P-450 enzymes during PAH and steroid biotransformation in C. elegans, and which can provide a valuable insight on such regulatory mechanisms in mammalian system.
This work was supported by a University of Łódź grant (no. 505/391).