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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Human Kidney Flavin-Containing Monooxygenases and Their Potential Roles in Cysteine S-Conjugate Metabolism and Nephrotoxicity

Renee J. Krause, Lawrence H. Lash and Adnan A. Elfarra
Journal of Pharmacology and Experimental Therapeutics January 2003, 304 (1) 185-191; DOI: https://doi.org/10.1124/jpet.102.042911

Abstract

The potential roles of human hepatic and renal flavin-containing monooxygenases (FMOs) in the metabolism of the cysteineS-conjugates S-allyl cysteine (SAC) andS-(1,2-dichlorovinyl)-l-cysteine (DCVC) were investigated. Incubations of human cDNA-expressed FMO1, FMO3, FMO4, and FMO5 with SAC resulted in detection of SAC sulfoxide, with FMO3 exhibiting approximately 3-, 4-, and 10-fold higher activity than FMO1, FMO4, and FMO5, respectively. DCVC sulfoxide formation was only detected with FMO3 and was 59-fold lower than SAC sulfoxide formation. Incubations of human liver microsomes with SAC or DCVC resulted in detection of the corresponding sulfoxides and provided evidence for the involvement of FMO3. Incubations of SAC or DCVC with human kidney microsomes, however, led only to the detection of SAC sulfoxide. Immunoblots with monospecific antibodies to FMO1, FMO3, and FMO5 in kidney microsomes from 26 humans showed that the average expression levels for FMO1, FMO3, and FMO5 were 5.8 ± 2.3, 0.5 ± 0.4, and 2.4 ± 1.4 pmol/mg (means ± S.D.), respectively. Interestingly, African-American kidney samples (n = 8) exhibited significantly higher FMO1 levels than Caucasian samples (n = 17), whereas no difference in expression level between males and females was observed with any of the examined FMO isoforms. Collectively, the results provide evidence for the expression of three FMO isoforms in the human kidney and show that the contribution of renal FMOs in cysteine S-conjugate metabolism is likely to vary depending upon the cysteineS-conjugate and the relative expression levels of the active FMOs.

Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze oxidation of sulfur-, selenium-, and nitrogen-containing compounds (Ziegler, 1993). Five active isoforms have been identified and are present in most mammalian tissues. However, species-, sex-, tissue-, and age-dependent differences exist in expression levels of these isoforms (Hines et al., 1994; Dolphin et al., 1996; Ripp et al., 1999a; Koukouritaki et al., 2002).

Human liver FMOs have been extensively characterized. FMO1 is present in liver of the human neonate, but its expression level is suppressed within the first few days of life and becomes undetectable thereafter (Koukouritaki et al., 2002). FMO2 mRNA was not detected in either adult or fetal liver samples (Dolphin et al., 1998). FMO3 is the predominant form expressed in adult human liver (Overby et al., 1997), and mutations in this isoform are responsible for trimethylaminuria, known as fish odor syndrome (Treacy et al., 1998). Transcripts for FMO4 have been detected in both the fetal and adult human liver, but the native protein has yet to be identified (Dolphin et al., 1996). FMO5 is also present in human liver at about one-third the amount of FMO3 (Overby et al., 1995).

In comparison with the liver, little is known about FMO expression in human kidney. FMO1 is expressed in adult human kidney (Hamman et al., 2000; Yeung et al., 2000). FMO1, FMO3, and FMO4 mRNA were detected in two adult human kidney samples, whereas FMO2 mRNA was detected in one of two adult kidney samples. Transcripts for FMO1 and FMO4 were detected in two fetal kidney samples (Dolphin et al., 1996, 1998). Renal FMOs were implicated in metabolism ofS-methyl-N,N-diethyl-dithiocarbamate, a metabolite of disulfiram (Pike et al., 2001). Metabolism of the anti-inflammatory drug sulindac by human kidney microsomes was correlated with relative FMO1 immunoquantitation in the same samples (Hamman et al., 2000). Collectively, these results suggest that FMO isoforms in addition to FMO1 may be expressed in human kidney and that the relative roles of these FMOs in renal metabolism of various chemicals and/or their metabolites may vary depending upon substrate selectivity and relative expression levels of the active FMO isoforms.

Glutathione conjugation is a major metabolic pathway for many chemicals and their electrophilic metabolites. Cysteine S-conjugates are formed during enterohepatic circulation and/or renal processing of the glutathione S-conjugates (Dekant et al., 1988; Hinchman and Ballatori, 1994; Elfarra, 1997). Cysteine S-conjugates can undergo S-oxidation to sulfoxides by FMOs present in human liver microsomes and rat and rabbit liver and kidney microsomes (Sausen and Elfarra, 1990; Sausen et al., 1993; Ripp et al., 1997,1999b). Rat and rabbit FMO isoforms exhibited selectivity in their abilities to oxidize cysteine S-conjugates (Duescher et al., 1994; Krause et al., 1996; Ripp et al., 1997). For example, among four recombinant rabbit FMOs (FMO1, FMO3, FMO4, and FMO5), FMO3 was the only isoform to convert the nephrotoxic cysteine S-conjugate of trichloroethyleneS-(1,2-dichlorovinyl)-l-cysteine (DCVC) to its sulfoxide DCVCS (Ripp et al., 1997). This pathway is significant because DCVCS can act as a Michael acceptor in the presence of cellular thiols (Sausen and Elfarra, 1991). DCVCS was a more potent nephrotoxin in rats than DCVC, both in vitro and in vivo (Lash et al., 1994). Because the precursor of DCVC,S-(1,2-dichlorovinyl)glutathione, was detected in the blood of humans exposed to trichloroethylene (Lash et al., 1999), DCVC may play a role in the renal toxicity of trichloroethylene in humans. DCVC induced cell death when incubated with human proximal tubular cells (Cummings and Lash, 2000; Lash et al., 2001). DCVC bioactivation in these cells may involve multiple mechanisms since partial protection from DCVC-induced necrosis was achieved using aminooxyacetic acid, a cysteine conjugate β-lyase inhibitor or methimazole, an alternative FMO substrate.

S-Allyl-l-cysteine (SAC) is a component of garlic and a known metabolite of allyl halides (Kaye et al., 1972). SAC has antioxidant properties and antiproliferative effects on melanoma and prostate carcinoma cells (Hatano et al., 1996;Ho et al., 2001; Takeyama et al., 1993; Pinto et al., 1997). SAC is metabolized by rat, rabbit, and human FMOs toS-allyl-l-cysteine sulfoxide (SACS) (Ripp et al., 1997, 1999a). Recently, we obtained evidence implicating FMOs in the in vivo metabolism of SAC in rats (Krause et al., 2002). Thus, the objectives of the present study were to determine whether SAC and DCVC are substrates for human FMOs, to characterize the metabolism of SAC and DCVC in human hepatic and renal microsomes and to quantitate expression levels of FMO1, FMO3, and FMO5 in human kidney samples.

Materials and Methods

Chemicals.

DCVC, DCVCS, and SACS were synthesized as previously described (Ripp et al., 1997). SAC was a generous gift from Wakunga Pharmaceutical of America (Mission Viejo, CA). NADPH, 2,4-dinitro-1-fluorobenzene, and aminooxyacetic acid were obtained from Sigma-Aldrich (St. Louis, MO). HPLC-grade acetonitrile was purchased from EM Science (Gibbstown, NJ). Primary rabbit antibodies to human FMO1, FMO3, and FMO5 peptides were purchased from BD Gentest (Woburn, MA). Microsomes containing cDNA-expressed human FMO1, FMO3, and FMO5 from baculovirus-transfected insect cells as well as control microsomes were also obtained from BD Gentest. The FAD content of the preparations was provided by the vendor. Escherichia coli membrane fractions containing cDNA-expressed FMO4 and expression vector alone were gifts from Dr. Richard M. Philpot (retired from National Institute of Environmental Health Sciences, Research Triangle Park, NC). Protein molecular weight standards (kaleidoscope-stained) were obtained from Bio-Rad (Hercules, CA). All other chemicals and reagents were of the highest quality commercially available.

Human Samples.

Kidney biopsy samples (n = 10) were obtained from the Human Tissue Resources Core of Wayne State University (Department of Pathology, Harper Hospital, Detroit, MI). Cadaver kidneys (n = 16) were procured by International Bioresearch Solutions (Pasadena, CA) for a total of 26 samples designated 1 to 26. Ages of the donors ranged from 16 to 81 years with a median age of 63.0 years. Seventeen donors were Caucasians, eight were African American, and one was of Hispanic descent. Microsomes were prepared as previously described (Lash et al., 1998). All microsomal samples were stored at −80°C until use. Protein concentrations of the human kidney samples were determined by the bicinchoninic acid method (Pierce Chemical, Rockford, IL), and the protein concentrations of the human liver and kidney samples used in the microsomal assays were determined by the method of Lowry et al. (1951). The human liver microsomal sample used in enzymatic studies was from a 44-year-old Caucasian female liver that was purchased from SRI International (Menlo Park, CA). The human kidney microsomal sample used in enzymatic studies was from a 68-year-old Caucasian female that was one of the samples that was used for immunoquantitation (number 12, Table1).

View this table:
Table 1

Quantitation of flavin-containing monooxygenases present in human kidney samples

cDNA-Expressed FMO and Human Microsomal Enzymatic Assays.

Typical reactions were carried out as previously described (Ripp et al., 1997). Reactions were carried out using microsomal fractions (50–100 μl containing 0.13–0.26 nmol flavin) and the β-lyase inhibitor, aminooxyacetic acid (1 mM). SAC incubations (10 mM) were stopped after 20 min with the addition of 0.5 ml of ethanol. DCVC (10 mM) reactions were stopped after 75 min with the addition of 0.5 ml of 0.75% perchloric acid. Samples for the analysis of DCVC and its corresponding sulfoxide were directly analyzed by HPLC at 220 nm, whereas the SAC samples were derivatized with 10% 2,4-dinitrofluorobenzene followed by HPLC analysis at 360 nm, as previously described (Ripp et al., 1997). The human liver microsomal incubations were carried out using 10 mM DCVC and 0.67 mg of protein from 0 to 75 min, whereas the human kidney microsomal incubations were carried out using 10 mM DCVC and 0.86 mg of protein at 75 min only due to the shortage of available protein. Human liver microsomes were incubated with 5 and 10 mM SAC for 0 to 40 min using 0.61 to 0.65 mg of protein, whereas the human kidney microsomal incubations were carried out using 5 and 10 mM SAC and 0.86 mg of protein at 40 min only. Recovery of SACS was also carried out at 40 min in the presence of both human liver and kidney microsomes and was >95%. Recovery of DCVCS was not carried out in the current study because of the limited amount of the human kidney microsomal sample but was found to be 71% in rabbit liver microsomes (Ripp et al., 1997) and 76% from rat liver microsomes (unpublished results).

HPLC Analyses.

HPLC analyses were performed using UV detection on a Gilson gradient controlled HPLC system and a Beckman 167 scanning absorbance detector on a Beckman ODS 5-μm reverse-phase C18 column (4.6 × 250 mm). The system was fitted with a 3-cm Brownlee guard column and a Bio-Rad AS100 Autosampler. The flowrate was 1 ml/min and 20-μl injections were made. Mobile phases were 1% acetonitrile, adjusted to pH 2.5 with trifluoroacetic acid on pump A, and 25% acetonitrile, pH 2.5, and 75% acetonitrile, pH 2.5, on pump B for DCVC and SAC analyses, respectively. The gradient for DCVC analysis of the cDNA-expressed FMO samples was as follows; the initial concentration was 20% B, at 3 min it increased to 60% B over 3.5 min, and at 11 min the gradient returned to 20% B over 3 min. Retention times for the two sulfoxide diastereomers were 3.9 and 4.1 min, respectively. The gradient for DCVC analysis for the human liver and kidney microsomal samples was as follows; the initial concentration was 5% B, which increased over 4 min to 15% B. Over the next 2.5 min, the gradient increased to 60% B where it was held for 4.5 min. The gradient then decreased to the initial concentration over 3 min where it was held for a total run time of 17 min. Retention times for the two sulfoxide diastereomers were 5.1 and 5.6 min, respectively, and the limit of detection was 22 pmol/20-μl injection. The gradient for SAC analysis of the cDNA-expressed FMO samples was as follows; the initial concentration was 30% B, at 5 min it increased to 80% B over 6 min, at 15 min the gradient decreased to the initial concentration over 3 min. Retention times of the SACS diastereomers were 6.5 and 7.3 min. For the analysis of the human microsomal samples, the initial and final concentration was 35% B, but the rest of the gradient remained the same. Retention times of SACS diastereomers were 6.5 and 7.3 min, respectively, and the detection limit was 57 pmol/20-μl injection. Enzymatically produced sulfoxides were quantified by comparing peak areas from the enzymatic reactions with peak areas from standard curves prepared in the same manner using synthetic sulfoxide standards (r2 > 0.99).

Immunoblotting.

All experiments contained as standards, one lane of prestained molecular weight markers and a standard curve of various amounts of cDNA-expressed FMOs. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using precast Criterion 12.5% resolving gels (Bio-Rad), followed by transfer to nitrocellulose membranes. For gels used to quantitate FMO1, 5 μg of kidney microsomes was loaded, whereas for gels used to quantitate FMO3 and FMO5, 20 μg of kidney microsomes was used. Nonspecific binding sites on the membranes were blocked by incubation in 5% nonfat dry milk in Tris buffered saline (25 mM Tris, pH 7.5, and 150 mM NaCl) for 1 h. Primary antibody to human FMO1, FMO3, or FMO5 prepared in rabbits was used at a 1:500 dilution and incubated for 1 h at room temperature. Goat anti-rabbit secondary antibody conjugated to peroxidase (Jackson Immunoresearch, West Grove, PA) was used at a 1:15,000 dilution. After incubation with secondary antibody, membranes were incubated with enhanced chemiluminescence immunodetection agents (Amersham Biosciences, Piscataway, NJ) and transferred to film. Immunoreactive proteins were quantified using Quantity One software (Bio-Rad), based on the optical density response of a standard curve generated with the same FMO standard on the same gel. Limits of detection are 5 fmol for FMO1 and FMO3 and 15 fmol for FMO5.

Statistics.

Statistical and correlation analyses were carried out using SigmaStat software (SPSS, Inc., Chicago, IL). Sex, race, and sample type comparisons were made using a two-samplet test with p < 0.05 set as the significance criterion. For the purpose of statistical analysis, values below the limits of detection were marked with a number in Table1 and were given an estimated numerical value. This value was based on the relative intensity of the detected band to the lowest standard within the standard curve limits.

Results

Incubations of SAC with the human cDNA-expressed FMO1, FMO3, FMO4, and FMO5 resulted in detection of SACS with all the isoforms (Table2). FMO3 produced the most SACS, followed by FMO1 and FMO4, whereas FMO5 produced smaller amounts of SACS. The reactions were stereoselective with FMO1 and FMO3 forming more diastereomer 2 (>90%), whereas the less active isoforms FMO4 and FMO5 produced the two diastereomers equally.

View this table:
Table 2

Comparison of SAC and DCVC oxidation by the various cDNA-expressed human FMO isoforms

The sulfoxidation of SAC (10 mM) with human liver microsomes was time-dependent. The results show that the reaction was linear between 5 to 40 min (Fig. 1); similar results were obtained when the reaction was carried out at a lower SAC concentration (5 mM; data not shown). The stereoselectivity observed in the human liver microsomal incubations was 88 to 100% diastereomer 2; the two SACS diastereomers exhibited similar recoveries (>95%) when incubated in the presence of human liver microsomes and NADPH for 40 min. Approximately twice the amount of SACS was detected in the incubations of human liver and kidney microsomes with SAC (10 mM) compared with the corresponding amounts detected at a lower SAC concentration (5 mM; Fig.2). The stereoselectivity of the diastereomers in the reaction with human kidney microsomes was nearly 90% diastereomer 2 for both concentrations. The activity with the human liver microsomal sample examined was nearly 10-fold higher than that observed with the kidney microsomal sample tested.

Figure 1
Figure 1

Cysteine S-conjugate metabolism by human liver microsomes. A, formation of SACS from 10 mM SAC over time. The stereoselectivity observed in these incubations ranged from 86 to 98%. B, formation of DCVCS from 10 mM DCVC over time. The stereoselectivity observed in these incubations ranged from 73 to 100%.

Figure 2
Figure 2

Metabolism of SAC by human liver (A) and kidney microsomes (B). The stereoselectivity was 90% and 88% at 5 and 10 mM, respectively. The human liver sample used was from a 44-year-old Caucasian female and the human kidney sample was from a 68-year-old Caucasian female.

DCVC was a substrate for human FMO3 only (Table 2). The human liver microsomal sample used for enzymatic assays produced DCVCS in a time-dependent manner; the stereoselectivity of the reaction was nearly 80% diastereomer 2 (Fig. 1). The small decrease in stereoselectivity of the DCVCS detected in human liver microsomes may be due to a small difference among the recoveries of the two DCVCS diastereomers. Incubations with human kidney microsomes failed to result in any detectable DCVCS.

Twenty-six human kidney microsomal samples were examined for the expression of FMO1, FMO3, and FMO5. Standard curves containing known amounts of human cDNA-expressed FMO1, FMO3, and FMO5 were used to quantitate the amount of the different isoforms in the human kidney microsomes. None of the FMOs examined cross-reacted with each other (Fig. 3). The proteins detected had similar mobilities as the cDNA-expressed standards (Fig. 3). All human samples had quantifiable amounts of FMO1, whereas not all samples had quantifiable FMO3 and FMO5. In fact, FMO1 is the predominantly expressed FMO isoform in all but one of the twenty-six samples examined (Table 1). The FMO1 expression level ranged from 3.2 to 11.5 pmol/mg of protein. FMO3 expression levels ranged from trace amounts to 1.3 pmol/mg of protein, whereas FMO5 levels were from trace amounts to 5.8 pmol/mg of protein. The mean expression levels for FMO1, FMO3, and FMO5 were 5.8 ± 2.3, 0.5 ± 0.4, and 2.4 ± 1.4 pmol/mg of protein (means ± S.D.), respectively (Table3). No significant difference in the expression levels of the individual isoforms was detected between the sexes. When the samples were analyzed by race, however, the African-American samples (n = 8) exhibited significantly higher amounts of FMO1 (7.7 ± 2.4 pmol/mg of protein; mean ± S.D.) than the Caucasian samples (n = 17; 5.0 ± 1.7 pmol/mg of protein; Table 3). No race-related differences in FMO3 or FMO5 expression levels were observed among the samples. When samples were analyzed by sample type, the cadaver samples (n = 16) exhibited significantly higher amounts of FMO5 (2.85 ± 1.38 pmol/mg of protein; mean ± S.D.) than the biopsy samples (1.58 ± 1.21 pmol/mg of protein; mean ± S.D.), whereas no differences were observed in FMO1 or FMO3 expression levels between cadaver and biopsy samples.

Figure 3
Figure 3

Immunoblots of human kidney samples. Lanes labeled A to I correspond to samples 1 to 9 in Table 2. A, reactivity with FMO1 antibody (Ab.); 5 μg of kidney microsomal protein was loaded. B, reactivity with FMO3 Ab.; 20 μg of kidney microsomal protein was loaded. C, reactivity with FMO5 Ab.; 20 μg of kidney microsomal protein was loaded. Molecular weight standards (MWS) and different amounts of recombinant human FMO1, FMO3, or FMO5 were loaded in the lanes preceding A to I to obtain standard curves. Standard FMO1, FMO3, and FMO5 were also loaded in the lanes after A to I to verify the specificity of the antibodies.

View this table:
Table 3

Sex and race-related expression of flavin-containing monooxygenases in human kidney samples

Discussion

The results presented in this article demonstrate that human FMOs can metabolize cysteine S-conjugates to yield the corresponding sulfoxides and that cysteine S-conjugates are selective in their interactions with human FMOs. The high stereoselectivity of SACS formation (>88% diastereomer 2) observed in the human liver microsomal incubations, along with the results obtained with the recombinant human FMOs, suggest major roles for FMO1 and/or FMO3 in the sulfoxidation reaction of SAC. Since previous studies have shown that FMO1 is not expressed in adult human liver, FMO3 is likely to be responsible for SAC sulfoxidation in human liver microsomes. Previously, our laboratory provided evidence that FMO3 is the primary enzyme involved in SAC sulfoxidation in rabbit liver microsomes from incubations with rabbit cDNA-expressed FMOs and that rabbit liver microsomal incubations in the presence and absence of catalase, superoxide dismutase, solubilizing agents, and cytochrome P450 inhibitors (Ripp et al., 1997). Ripp et al. (1999) also determined theKm value of human FMO3 to be 5.0 ± 0.8 mM (mean ± S.D). Because we observed nearly twice as much activity in both the liver and kidney samples using 10 mM SAC compared with 5 mM SAC, the enzymes may not yet be saturated. This suggests that the Km for this reaction is high and that at higher SAC concentrations, higher activity may be observed. It was not practical to determine the Kmvalues for SAC and DCVC because of the limited supply of recombinant FMOs available for the present study and because of the solubility problems of high concentrations of DCVC at physiological conditions (pH 7.4, 37°C).

The extent of the stereoselectivity observed in the reaction with the human kidney microsomes suggests that either FMO1 or FMO3 or both are responsible for the majority of the sulfoxidation observed with SAC. Based upon the FMO expression data presented in Table 2, it is likely that FMO1 is the major contributor to SACS formation in human kidney microsomes. Cysteine S-conjugates that are selective substrates for FMO1, such asS-benzyl-l-cysteine (Krause et al., 1996; Ripp et al., 1997), may also be metabolized in the human kidney to their corresponding sulfoxides. Our inability to detect DCVCS with the human kidney microsomal sample used for enzymatic assays in the present study was not unexpected. As shown in Tables 1 and 2, DCVC is only a substrate for FMO3, which is expressed at low levels in the human kidney compared with the liver, and FMO3 exhibited much lower activity with DCVC than with SAC. Freshly isolated rat kidney distal tubular cells were previously shown to exhibit higher FMO activity withS-benzyl-l-cysteine than rat kidney proximal tubular cells (Lash et al., 1994); in future studies, microsomes from isolated proximal and distal tubular cells will be examined for FMO expression and activities since FMO3 may not be evenly expressed in these cells, and this could be an important determinant of DCVC bioactivation and toxicity in human kidney cells. Nonetheless, because of its potent nephrotoxicity in comparison with DCVC, any DCVCS that may be formed in kidney cells or that could be translocated to kidney cells through the circulation after being formed in the liver could play an important role in DCVC nephrotoxicity. Evidence for this hypothesis is provided by the previous finding that DCVCS administration to rats caused selective nephrotoxicity that was preceded by significant depletion of renal reduced nonprotein thiol concentrations (Sausen and Elfarra, 1991; Lash et al., 1994).

The results presented in this article clearly show that in addition to FMO1, the FMO3 and FMO5 proteins are also present in the human kidney and present the first quantitative data on the expression levels of FMO1, FMO3 and FMO5 at the protein level in the same kidney sample from a relatively large number of subjects (n = 26). A previous study examined FMO1 expression in four human kidney samples and found the average content of FMO1 to be 47 ± 9 pmol/mg of protein (Yeung et al., 2000). This is higher than the mean value for FMO1 expression in our study (6 ± 2 pmol/mg of protein). Several factors may have contributed to the different results between the two studies. For example, Yeung et al. (2000) used an antibody prepared against minipig liver FMO1, whereas we used an antibody that was prepared against a peptide sequence in human FMO1. Some variability in the results may also be due to differences in sample preparation and/or storage conditions between the two studies. FMO1 was expressed at a higher level than either FMO3 or FMO5 in all but one of the 26 samples. Statistical analyses comparing cadaver versus biopsy-derived samples showed a significantly higher amount of FMO5 in the cadaver samples than the biopsy samples. No statistical significance was observed in the expression of FMO1 or FMO3 among cadaver and biopsy samples. Collectively, these results suggest that there was little or no degradation of FMO expression in the cadaver samples tested. Our results show a 3- to 4-fold variation in FMO1 expression levels among the 26 samples examined, consistent with the results obtained by Hamman et al. (2000) in which an approximately 5-fold variation in the relative FMO1 expression level was observed in 13 human kidney samples using an antibody prepared against rabbit liver FMO1. Unfortunately, no quantitation was provided in the study by Hamman et al. (2000), as only the relative amounts of FMO1 expression were reported in that study.

Our results show a 5- to 6-fold variation in FMO3 expression and a 10- to 20-fold variation in FMO5 expression levels among the 26 human kidney samples examined. In most samples, the levels of FMO3 and FMO5 present were lower than the FMO1 level present in the same sample. A 10-fold variation in FMO3 content in human liver was observed by Overby et al. (1997), whereas Koukouritaki et al. (2002) reported a 2- to 20-fold variation based on the age of the person from which the sample was obtained.

A significantly higher level of FMO1 expression was observed in the African-American kidney samples compared with the Caucasian samples. Although the molecular basis for this observation is presently unclear, yin yang 1 (YY1) and hepatic nuclear factors 1 and 4 have been shown to be involved in regulation of FMO1 expression (Luo and Hines, 2001). FMO1 promoter activity can be negatively regulated by YY1. Hines and coworkers (personal communication) have observed a single nucleotide polymorphism (SNP) in the YY1 site that occurs at a significant frequency (25%) in the Hispanic-American samples examined (n = 60). Although a definite frequency in the Caucasian population has yet to be determined, the SNP also appears to occur with frequency in this population. The SNP does not appear, however, to be present or occurs at a low frequency in the African-American samples (n = 100) examined (Dr. R. Hines, personal communication; Medical College of Wisconsin, Milwaukee, WI). This lack of the SNP observed in the African-American population may explain the significantly higher amount of FMO1 observed in our African-American samples. Unfortunately, whole tissue homogenates from the kidney samples used in our studies are not presently available to test this hypothesis. Since several drugs and cysteineS-conjugates can be metabolized by FMO1, race differences in FMO1 expression in the kidney may be of pharmacological and/or toxicological significance. Ethnic differences have previously been demonstrated in FMO2 expression; functional FMO2 does not appear to be present in Caucasians (Dolphin et al., 1998); however, approximately 25% of African Americans have functional full-length FMO2 (Whetstine et al., 2000).

In summary, the results presented in this article suggest a role for FMO1, FMO3, and FMO5 in cysteine S-conjugate metabolism in the human kidney. The relative contributions of these FMOs in cysteineS-conjugate metabolism in the kidney are likely to vary depending upon the chemical structure of the cysteineS-conjugate and the relative expression levels of the active FMOs. Although we have used 26 kidney samples in the present study, future use of additional samples could enhance the significance of our results. In this regard, future studies should examine the molecular basis for the observed race-related differences in FMO1 expression in the human kidney, as these differences may have significant physiological, pharmacological, and/or toxicological implications.

Acknowledgments

We acknowledge the technical assistance of Kathryn Stettler and Carrie Klumb.

Footnotes

  • This research was supported by Grant DK44295 from the National Institute of Diabetes, Digestive, and Kidney Diseases (A.A.E.) and by Grant ES08828 from the National Institute for Environmental Health Sciences (L.H.L.). A preliminary report of these results was presented at the Federation for the American Societies of Experimental Biology meeting held in New Orleans, LA on April 21–25, 2002.

  • DOI: 10.1124/jpet.102.042911

  • Abbreviations:
    FMOs
    flavin-containing monooxygenases
    DCVC
    S-(1,2-dichlorovinyl)-l-cysteine
    DCVCS
    S-(1,2-dichlorovinyl)-l-cysteine sulfoxide
    SAC
    S-allyl-l-cysteine
    SACS
    S-allyl-l-cysteine sulfoxide
    HPLC
    high-performance liquid chromatography
    YY1
    ying yang 1
    SNP
    single nucleotide polymorphism
    • Received August 8, 2002.
    • Accepted September 25, 2002.

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Human Kidney Flavin-Containing Monooxygenases and Their Potential Roles in Cysteine S-Conjugate Metabolism and Nephrotoxicity

Renee J. Krause, Lawrence H. Lash and Adnan A. Elfarra
Journal of Pharmacology and Experimental Therapeutics January 1, 2003, 304 (1) 185-191; DOI: https://doi.org/10.1124/jpet.102.042911
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