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NDT Advance Access originally published online on May 21, 2008
Nephrology Dialysis Transplantation 2008 23(10):3074-3081; doi:10.1093/ndt/gfn262
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Induction of P450 1A by 3-methylcholanthrene protects mice from aristolochic acid-I-induced acute renal injury

Xiang Xue1,2*, Ying Xiao1,2*, Hongli Zhu3, Hui Wang1, Yongzhen Liu1, Tianpei Xie3 and Jin Ren1

1 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences 2 Graduate School of the Chinese Academy of Sciences 3 Shanghai TenGen Biomedical Company, Shanghai, China

Jin Ren, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhang Jiang Hi-Tech Park, Shanghai 201203, China. Fax: +86-21-50806031; E-mail: cdser_simm{at}mail.shcnc.ac.cn



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 
Background. Cytochrome P450 1A, an enzyme known to metabolize polycyclic aromatic hydrocarbons (PAHs), participates in the metabolism of aristolochic acid I (AAI) in liver and kidney microsomes isolated from humans and rodents. This study was designed to investigate whether P450 1A plays a role in AAI-induced renal injury in C57BL/6 mice.

Methods. Separate groups of mice were given AAI (10 mg/kg and 20 mg/kg) or pretreatment with 3-methylcholanthrene (3-MC, an agent known to induce P450 1A expression in many species including rodents) at 60 mg/kg given at 24 h before AAI injection. Renal function and histopathology were determined at the 3rd day following the high dose of AAI and at the 14th day following the low dose of AAI treatment. For both doses, we determined in vivo AAI clearances and pharmacokinetic parameters. We also determined in vitro P450 1A1/2 activity and the ability of liver microsomes from 3-MC-treated and vehicle-treated mice to metabolize AAI. Finally, the effect of 3-MC on protein levels of P450 1A1/2 in both liver and kidney was measured by western blotting.

Results. Pretreatment with 3-MC greatly protected mice against renal failure induced by AAI. In vivo AAI clearance was more rapid in 3-MC-pretreated mice than in the vehicle-pretreated mice. In addition, the P450 1A1/2 activity and the ability to metabolize AAI in hepatic microsomes isolated from 3-MC-treated mice were much greater than in vehicle-treated mice. Western blotting showed that protein levels of hepatic P450 1A1/2 were greatly increased in 3-MC-treated mice than in vehicle-treated mice.

Conclusion. These results demonstrated that the induction of hepatic P450 1A1/2 protected against AAI-induced kidney injury through faster in vivo clearance of AAI and suggested an important role for hepatic P450s in the detoxification of AAI-induced renal injury.

Keywords: aristolochic acid nephropathy; P450 1A; 3-methylcholanthrene



   Introduction
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 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 
Aristolochic acid-associated nephropathy (AAN) is characterized by hypocellular interstitial fibrosis, intimal thickening of interlobular and afferent arterioles, with glomerular sparing or mild sclerosis [1,2]. Since the first reported case in Belgium, many instances of AAN in adults have been reported throughout the world [3,4,5]. In all cases of AAN, patients had taken Chinese herbs containing aristolochic acid (AA) [6]. The well-known nephrotoxicity of AA had been established in animal models treated with AA or AA-containing herbs [7]. Balkan endemic nephropathy (BEN) appears similar to AAN on both morphological and clinical grounds [2], and a recent report provided direct evidence that AA may be a significant risk factor for BEN [8,9].

AA is a mixture of derivatives of 3,4-methylenedioxy-10-nitro-1-phenanthrenecarboxylic acid and has been used as an anti-inflammatory agent in Germany [10]. There are at least six components in AA, among which arisotolochic acid I (AAI) is the main ingredient and the most cytotoxic [11]. Metabolic studies examining AA in different species including humans showed that aristolactams (AL) are the major metabolites found in excreta after nitro reduction [12]. In both rat and human hepatic microsomes, the in vitro activation of AA was mostly attributed to cytochrome P450 1A1/2 [13], and the major metabolite was AAIa under aerobic conditions in rat liver 9000 g supernatant [14]. Although the actions of P450s and other enzymes to form AA-DNA adducts have been widely studied in AA carcinogenesis experiments [15,16], little research has focused on the in vivo mechanisms of AA-induced kidney toxicity, especially at the level of enzymes and their in vivo metabolic effects. Thus, the role that P450s play in AA-induced kidney toxicity and whether in vivo induction of P450 1A, an important P450 isoform involved in the metabolism of AA, will affect AA-induced kidney toxicity are largely unknown.

It has been reported that 3-methylcholanthrene (3-MC), a potent inducer of aryl hydrocarbon hydroxylase activity [17], induces P450 1A expression at the level of both mRNA and protein in many species, including mice [18–20]. Thus, the aim of this study was to investigate the effect of P450 1A induction by 3-MC on AAI-induced renal injury in C57BL/6 mice. We found that pretreatment of the mice with 3-MC greatly reduced the kidney toxicity induced by AAI. These findings suggest an important role for hepatic P450s in the detoxification of AAI-induced renal toxicity.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Supplementary data
 References
 
Reagents
Aristolochic acid I (AAI), 3-methylcholanthrene (3-MC), 7-ehtoxyresorufin and 7-methoxyresorufin were purchased from Sigma Chemical Co. (St Louis, MO, USA). The standard for aristolochic acid Ia (AAIa) was a generous gift from Dr Minghua Xu. The L-Type Creatinine F kit and L-Type UN kit were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) Rabbit monoclonal antibodies to GAPDH were from Cell Signaling Technology, Inc. (Danvers, MA, USA), mouse monoclonal antibodies to cytochrome P450 1A1/2 and rabbit polyclonal antibodies to cytochrome P450 reductase (CPR) were from Abcam (Cambridge, UK), and sheep polyclonal antibodies to P450 1A2 were from Chemicon (Temecula, CA, USA). All other reagents were from Sigma and were of analytical grade.

Animal protocols
Animal-use protocols were approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica (Shanghai, China). Two-month-old male C57BL/6 mice were used in the studies. Animals were maintained at 22°C with a 12-h on and 12-h off light cycle. 3-MC (60 mg/kg, in corn oil) pretreatment was given 24 h before AAI injection, whereas animals in the control groups received vehicle. For AAI clearance experiments, the animals were given a single intraperitoneal (i.p.) injection of AAI (10 mg/kg and 20 mg/kg, in warm saline), and blood samples were collected by tail-bleeding at various times after the injection. For serum blood urea nitrogen (BUN) and creatinine determinations as well as for histological examinations, blood and tissue samples were collected at 14 days or 3 days after a single i.p. injection of AAI (10 mg/kg or 20 mg/kg, respectively).

Determination of serum BUN and creatinine levels
Serum creatinine and BUN levels were determined by the L-Type Creatinine F kit and the L-Type UN kit according to vendor instructions using a Hitachi-7080 biochemical analyzer.

Histopathological examination
The mice were killed at various times after AAI injection. Blood samples were drawn by cardiac puncture, and livers and kidneys were then promptly collected and fixed in 10% formalin for 24 h. The tissues were sectioned at 2–3 mm and fixed in 10% formalin for an additional 24 h. After dehydration through a series of alcohols, tissues were cleared in xylene and impregnated with paraffin wax prior to embedding in blocks of paraffin wax. Sections were then cut at 3–4 µM and floated onto glass slides, baked at 56°C for 30 min and stained with haematoxylin-eosin (HE) and Masson's trichrome staining.

Determination of blood AAI and its major metabolite levels
Blood samples (15 µl each) were collected in heparin-coated capillary tubes and were mixed with an equal volume of saline. The samples were spun at 4000 g for 5 min at 4°C. The supernatant fractions were mixed with one-half volume of methanol and spun again at 14 000 g for 5 min to remove the precipitates. Aliquots of the final supernatant were analysed for AAI concentration on a high-performance liquid chromatograph according to the method described by Krumbiegel [12]; typical HPLC chromatograms are shown as supplementary data. Pharmacokinetic parameters were calculated using the WinNonLin software, version 3.1.

Isolation of microsomes
Hepatic tissues from three mice were homogenized in three volumes of 0.15 M KCl and centrifuged at 9000 g for 20 min. Microsomes were obtained by centrifugation of the supernatant at 100 000 g for 1 h and resuspension of the resulting pellets in a buffer containing 0.25M sucrose and 0.05M Tris buffer at pH 7.5. All steps were performed at 4°C. Protein concentrations were determined using a BCATM protein assay kit (Pierce, IL, USA).

Measurement of ethoxyresorufin and methoxyresorufin O-dealkylase activity
O-dealkylase activity was determined by the formation of 7-ethoxyresorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation (MROD) as described by Burke [21]. Liver microsomes (2 mg/ml protein) were preincubated with a NADPH generating system (0.3 mM NADP+, 0.4 mM glucose 6-phosphate, 1.2 mM MgCl2 and 0.4 U/ml glucose-6-phosphate dehydrogenase) in 0.1M phosphate buffer, pH 7.4 (total volume 0.25 ml) at 37°C for 5 min. Reactions were initiated with 7-ethoxy- or 7-methoxyresorufin (10 µM) and terminated at 120 min by adding 125 µl ice-cold acetonitrile. Samples were allowed to precipitate on ice and centrifuged for 10 min at 14 000 g. Supernatants were analysed spectrofluorometrically for product (resorufin) at an excitation of 545 nm and emission of 590 nm. Experimental values were extrapolated using a standard curve in the linear range.

Microsome metabolism
Incubations were carried out at 37°C for 2 h in tubes containing 20 µg/ml AAI dissolved in 25 µl DMSO, 2 mg/ml microsomal protein and an NADPH generating system (5 mM glucose-6-phosphate, 3 mM MgCl2, 1 mM NADP+ and 1.5 units of glucose-6-phosphate dehydrogenase) in 100 mM sodium phosphate, pH 7.4, in a total volume of 250 µl. The reaction was initiated by an addition of the NADPH generating system and stopped by the addition of 125 µl ice-cold acetonitrile. The precipitate was removed by centrifugation at 14 000 g for 10 min. AAI concentration in the supernatant was detected on a high-performance liquid chromatograph according to the method as described [12].

Western blotting
Proteins were separated by electrophoreses on a SDS-10% polyacrylamide gel and then transferred to the PVDF membrane (Amersham). The membranes were blocked in 5% skim milk powder in PBS before overnight incubation with a monoclonal rabbit anti-mouse GAPDH (1:1000) or anti-mouse cytochrome P450 1A1/2 (1:500) or polyclonal rabbit anti-mouse CPR (1:1000) or sheep anti-rat P450 1A2 (1:1000) antibody. Proteins were visualized using an ECL detection system (Amersham, NJ, USA) and a Kodak Image Station (Kodak, CT, USA).

Statistical analysis
Data are expressed as means ± SD. One-way ANOVA and Student's t-test were used for statistical analysis, with P < 0.05 accepted as significant difference.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Supplementary data
 References
 
AAI-induced renal fibrosis in C57BL/6 mice
To establish the animal model for AAN in our laboratory, we first performed acute toxicity experiments to determine appropriate doses and sacrifice times. We found that the median lethal dose (LD50) was ~37 mg/kg for male C57BL/6 mice (data not shown), and that AAI induced a rapid progressive tubulointerstitial fibrosis in mice kidneys at 14 days after single i.p. injections at doses of 10 and 20 mg/kg. Figure 1 shows the characteristics of mice kidneys at 14 days after 10 mg/kg AAI that included hypocelluar interstitial fibrosis, tubular atrophy, limited interstitial infiltrates and relatively mild glomerular changes, which are characteristics of this form of nephropathy [22]. These results suggest that a single 10 mg/kg AAI i.p. injection can induce AAN in an animal model of C57BL/6 mice.


Figure 1
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  		Fig. 1 Representative photomicrographs of renal cortex. (A) No abnormalities were found in control mice. (B) Severe tubular atrophy and interstitial fibrosis (arrows) were observed in mice at 14 days following AAI (10 mg/kg) (Masson's trichrome staining. Original magnifications x400).

 
3-MC-pretreatment prevented renal injury induced by AAI
To assess the effect of 3-MC on renal injury induced by AAI, we first examined BUN, serum creatinine and kidney histopathology. As shown in Figure 2, single injections of AAI at 10 or 20 mg/kg greatly elevated both BUN and creatinine levels. Pretreatment with 3-MC blocked these elevations induced by 10 mg/kg AAI at the 14th day post-injection (Figure 2A and B). At the 3rd day following injection of 20 mg/kg AAI, both the 3-MC-pretreated and vehicle-pretreated groups had higher BUN and creatinine levels than controls; however, these elevations were mild in the 3-MC-pretreated group (Figure 2C and D). Histopathological examination of the kidneys showed a normal appearance in control mice (Figure 3A and D). At 14 days following 10 mg/kg AAI, vehicle-pretreated mice had severe tubulointerstitial lesions, hyperplasia and fibrosis (Figure 3B), which contrasted with a loss of tubular epithelia with slight interstitial hyperplasia and fibrosis in 3-MC-pretreated mice (Figure 3C). At the 3rd day after 20 mg/kg AAI, vehicle-pretreated mice exhibited a large area of tubular lesions, loss of tubular brush border, necrotic cells and cellular fragments in the lumen of proximal tubules (Figure 3E), while 3-MC-pretreated mice showed the same tubular lesions but within a much smaller area (Figure 3F). We graded lesion severity in these groups of mice and found that the severity was greater in vehicle-pretreated mice than in 3-MC-pretreated mice at both doses of AAI Table (1). These results suggest that 3-MC pretreatment may reduce the renal injury induced by AAI.


Figure 2
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  		Fig. 2 Effect of 3-MC on blood urea nitrogen (BUN) and serum creatinine. (A and B) Mice treated with 10 mg/kg AAI and assayed at 14 days after the treatment. (C and D) Mice treated with 20 mg/kg AAI and assayed at 3 days after the treatment. Values are means ± SD. n = 5 for each group. **P < 0.01, compared with control group.

 

Figure 3
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  		Fig. 3 Histological examination of mice kidneys. (A) Control mice, (B) 10 mg/kg AAI-treated mice and (C) 3-MC pretreated 10 mg/kg AAI-treated mice, all at the 14th day following AAI treatment; (D) control mice, (E) 20 mg/kg AAI-treated mice and (F) 3-MC pretreated 20 mg/kg AAI-treated mice, all at the 3rd day following AAI. Arrows indicate the main area of injury. (H&E stain, original magnifications x200, x400.)

 

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  		Table 1 Extent of kidney injury in vehicle-pretreated and MC-pretreated mice

 
AAI clearance in 3-MC-pretreated mice was more rapid than in vehicle-pretreated mice
We next examined whether there are differences in AAI metabolism between vehicle-pretreated mice and 3-MC-pretreated mice. As shown in Figure 4, blood levels of AAI at 5–60 min after injection were markedly reduced in 3-MC-pretreated mice compared with levels in the vehicle-pretreated mice (P < 0.01). Blood AAI level began to noticeably decrease in 3-MC-pretreated mice at 5 min after AAI injection, while in vehicle-treated mice it peaked at 15 min after AAI injection. The pharmacokinetic parameters, shown in Table 2, demonstrated that Tpeak, Cmax, AUC and CL/Fs in 3-MC-pretreated mice were significantly different than the values in vehicle-pretreated mice. We also examined major metabolites of AAI in serum (Figure 4C and D), of which only AAIa was detectable. Serum AAIa levels were lower in 3-MC-pretreated mice than in vehicle-pretreated mice, and the time to initial decrease was earlier in 3-MC-pretreated mice than in vehicle-pretreated mice. These results suggest that 3-MC pretreatment accelerated the in vivo clearance of AAI, and that the major metabolite generated in vivo is AAIa.


Figure 4
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  		Fig. 4 Serum clearance of AAI, its major metabolite (AAIa) in C57BL/6 mice. Vehicle-pretreated and 3-MC-pretreated mice were given a single i.p. injection of 10 mg/kg (A, C) or 20 mg/kg (B, D) AAI, and blood samples were collected after injection for the determination of serum AAI concentration and for the quantity of the major metabolite. AAIa is expressed as the peak area (mAU min) of the AAIa peak in the HPLC chromatogram. Values are means ± SD. n = 5 for each group. **P < 0.01, compared with 3-MC-pretreated group.

 

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  		Table 2 Pharmacokinetic parameters of AAI in vehicle-pretreated and 3-MC-pretreated mice

 
Enzyme activities demonstrated by EROD and MROD assays
Since P450s play a critical role in metabolizing AAI [14], we tested the effects of 3-MC on hepatic microsomal P450 activities. EROD and MROD assays were used to determine P450 1A1 and 1A2 activities. We found that 1A1 (Figure 5A) and 1A2 (Figure 5B) activities in hepatic microsomes isolated from 3-MC-pretreated mice were 70 times and 8 times, respectively, higher than in vehicle-pretreated mice. These results suggest that pretreatment with 3-MC increased the enzyme activity of P450 1A in the liver of the mice.


Figure 5
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  		Fig. 5 Effect of 3-MC on hepatic O-dealkylations of ethoxyresorufin (EROD) and methoxyresorufin (MROD) in vehicle- and 3-MC-pretreated mice. (A) EROD assay for P450 1A1 activity. (B) MROD assay for P450 1A2 activity. Values are means ± SD. n = 5 for each group. **P < 0.01, compared with vehicle-pretreated mice.

 
In vitro metabolism of AAI by microsomes
To further determine whether AAI metabolism was affected by 3-MC, we examined the metabolic capacity of hepatic microsomes isolated from vehicle- or 3-MC-pretreated mice to metabolize AAI. After incubation for 2 h, AAI concentrations in microsomes isolated from vehicle-treated mice remained ~70% of the initial concentration; whereas it was ~50% of the initial concentration in 3-MC-pretreated mice (Figure 6A). We also examined the major metabolite AAIa generated during incubation, which was quantified from the peak area of the AAIa peak in the HPLC chromatogram (Figure 6B). We found that AAIa levels were significantly greater in microsomes from 3-MC-pretreated mice than that from vehicle-pretreated mice. These results suggest that 3-MC treatment increased the capacity to metabolize AAI in the liver of the mice.


Figure 6
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  		Fig. 6 Metabolism of AAI in microsomes isolated from the liver of vehicle- and 3-MC-pretreated mice. (A) Relative concentration of AAI after the incubation of microsomes. (B) Quantity of the major metabolite generated in the incubation. AAIa is expressed as the peak area (mAU min) of the AAIa peak in the HPLC chromatogram. Values are means ± SD. n = 5 for each group. **P < 0.01, compared with control group.

 
P450 1A1/2 and CPR expression in liver and kidney of vehicle- and 3-MC-pretreated mice
To determine whether 3-MC induces P450 1A1/2 expression in liver to then enhance the metabolism of AAI, we examined protein levels of these enzymes in the mice (Figure 7). Western blot analysis revealed that protein levels of P450 1A1/2, and especially P450 1A2, were greatly increased in 3-MC-pretreated mice compared with the levels in control mice. CPR, the obligate redox partner for all microsomal P450s, was slightly increased in the liver of 3-MC-pretreated mice. These results support the notion that 3-MC induces hepatic P450 1A1/2 expression to then facilitate AAI clearance from the body. It has been reported that P450s in kidney also can metabolize AAI. Because of this, we also examined whether 3-MC treatment affected the expression of P450 in kidney. We found that the expressions of P450 1A2 and CPR were almost unchanged in 3-MC treated mice compared with controls. These results suggest that 3-MC treatment increased the expression of P450 1A1/2 in liver but not in kidney, which may have contributed to the faster clearance of AAI and the reduced renal toxicity in these mice.


Figure 7
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  		Fig. 7 Western blot analysis of cytoplasmic preparations from liver and kidney of vehicle- and 3-MC-pretreated mice. L indicates liver; K indicates kidney. Each lane contained a tissue pool from two mice.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Supplementary data
 References
 
AA intake has been associated with the development of AAN and Balkan endemic nephropathy (BEN) [8]. The mechanisms of AA-induced diseases have been studied both in vitro and in vivo [23,24]. In spite of many in vitro studies examining AA metabolism and mechanisms of its carcinogenesis [13,16], there has been little in vivo investigation on the mechanism by which AA induces renal toxicity. Furthermore, it remains unknown which organs participate in the in vivo activation of AA and whether AA or its metabolite contributes to the renal toxicity. In the present study, we report a protective effect of 3-MC, a potent inducer of P450 1A, against kidney injury induced by AAI, the major component of AA. Our results provide a possible explanation for the in vivo mechanism by which AAI induces renal toxicity and suggest that hepatic P450 1A1/2 contributes to the clearance of AAI, probably by a detoxification process acting against AAI-induced kidney injury.

Here we showed a protective effect of 3-MC, as demonstrated by the renal function analysis and histological examination, and then studied possible mechanisms. While examining the in vitro metabolism of AAI, we found that liver microsomes isolated from 3-MC-pretreated mice had greater activity than liver microsomes isolated from vehicle-pretreated mice. This occurred in parallel with 3-MC-induced elevated activities of hepatic P450 1A1 and 1A2, which was probably a result of the increased expression at the protein level. As a result, the in vivo clearance of AAI was faster in 3-MC-pretreated mice than in vehicle-pretreated mice, as demonstrated by serum AAI concentrations and pharmacokinetic parameters. In addition, we found that the major metabolite generated in vivo is AAIa, which is in agreement with previous in vitro studies using rat homogenate and which was authenticated by the AAIa standard. AAIa levels were lower in 3-MC-pretreated mice, and this was possibly because of lower AAI bioavailability. AAIa can be transformed into aristolactam Ia (ALIa) [25], which explains why the principal metabolite of AAI found in urine and faeces in rats was ALIa [12]. Both AAIa and ALIa are much less cytotoxic and mutagenic than AAI [11,14], and it is clear that the metabolites generated in liver were less toxic than AAI. Thus, the in vivo toxicity of AAI was correlated with its serum concentration, and a more rapid clearance of AAI may lead to less accumulation, resulting in less kidney toxicity.

In vitro metabolic studies using inhibitors of various enzymes and recombinant enzymes that detected and quantified specific AA-DNA adducts showed that prostaglandin H synthase (PHS) also contributes to the activation of AAI in the kidney [26]. Although a possible role for PHS in the kidney activation of AAI could not be ruled out, the finding that protein levels of P450 1A1/2 remained unchanged in kidney suggested that the kidneys may play a less important role in metabolizing AAI. Moreover, there are no reports that 3-MC affects the expression and activity of PHS or other related enzymes. Although 3-MC also induces P450 1A in intestine and seminal vesicles in rat, the increase is very mild [27]. Therefore, it is unlikely that 3-MC will influence the metabolism of AAI in the kidney or other organs, and if it does this would be to a much smaller extent.

The in vivo expression and in vitro metabolism findings for P450 1A in the current experiment are consistent with a previous in vitro study showing that P450 1A1/2, and especially 1A2, is responsible for most of AA metabolism [13]. High levels of hepatic P450 1A1/2 proteins lead to rapid metabolism of AA in the liver, which subsequently result in less toxicity in the kidney. There is a variability of P450 1A in human beings [28], such that people with low expression of P450 1A face a high risk of AAN and BEN, which may explain why not all people exposed to AA subsequently develop AAN or BEN. An assay for variability of P450 1A in populations that have potential for exposure to AA may provide a means to predict and prevent the risk of AAN and BEN.

AA is activated and subsequently reacts with cellular proteins and DNA, producing DNA adducts in both kidney and liver [29]. Although the liver is the major organ for biotransformation of xenobiotics, AA induces tumours only in the kidney. In addition, a case report showing Aristolochia-related urinary tract cancer without significant renal failure suggested a dissociation between carcinogenicity and nephrotoxicity of AA [30]. In the present study we focused on the in vivo mechanisms of nephrotoxicity of AA by investigating the in vivo effects of major enzymes responsible for AA metabolism on AA-induced kidney toxicity. Understanding the underlying mechanisms of tissue-specific carcinogenicity of AA will require further study.

In conclusion, we have shown that inducement of P450 1A by 3-MC can increase the in vivo clearance of AAI by inducing enzymes involved in AAI metabolism, resulting in the reduced AAI toxicity in the kidney. These results suggest that hepatic P450s play a detoxification role in AAI-induced renal injury. A mouse model in which hepatic P450s are specifically suppressed will be useful to provide further evidence that hepatic P450s play a key role in AA-induced kidney toxicity. Future studies will also be needed to identify AAI metabolites generated in the liver and kidney of mice.



   Supplementary data
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data is available online at http://ndt. oxfordjournals.org



   Acknowledgments
 
We thank Professors Yizheng Wang and Jun Gu for reading the manuscript and appreciate the excellent technical assistance of Hua Sheng, Henglei Lu and Jingjing Chen. This research was supported by the National Grand Fundamental Research 973 Program of China (No. 2006CB504700).

Conflict of interest statement. None declared.



   Notes
 
* Both authors contributed equally to this work. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 

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Received for publication: 11.10.07
Accepted in revised form: 16. 4.08


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