Acute cadmium chloride administration induces ...

Toxicology Letters 148 (2004) 199–210

Acute cadmium chloride administration induces hepatic and renal CYP2A5 mRNA, protein and activity in the mouse: involvement of transcription factor NRF2 A’edah Abu-Bakar a,∗ , Soisungwan Satarug a , Geoffrey C. Marks d , Matti A. Lang c , Michael R. Moore a,b a

c

National Research Centre for Environmental Toxicology, University of Queensland, 39 Kessels Road, Coopers Plains, 4108 Qld, Brisbane, Australia b Queensland Health Scientific Services, Qld, Brisbane, Australia Department of Pharmaceutical Biosciences, Division of Biochemistry, Uppsala University, Uppsala, Sweden d School of Population Health, University of Queensland, Medical School, Qld, Brisbane, Australia Received 3 October 2003; received in revised form 30 October 2003; accepted 31 October 2003

Abstract Modulation of the cytochrome P450 (CYP) monooxygenase system by cadmium was investigated in male, adult DBA/2J mice treated with a single dose (16 ␮mol/kg body weight, i.p.) of cadmium chloride (CdCl2 ). Total CYP content of liver and kidney microsomes decreased maximally (56% and 85%, respectively) 24 and 18 h, respectively, after CdCl2 treatment. Progressive increases of hepatic coumarin 7-hydroxylase (COH) activity; indicative of CYP2A5 activity, relative to the total CYP content were seen at 8 h (2-fold), 12 h (3-fold), 18 h (12-fold), and 24 h (15-fold). Similar changes were seen in the kidney. Liver and kidney CYP2A5 mRNA levels increased maximally 12 and 4 h after treatment and decreased to almost half 6 h later. In contrast, kidney and liver CYP2A5 protein levels increased maximally at 18 and 24 h. The CYP2A5 mRNA levels in the kidney and liver increased after Cd treatment in Nrf2 +/+ but not in Nrf2 −/− mouse. This study demonstrates that hepatic and kidney CYP2A5 is upregulated by cadmium with a somewhat faster response in the kidney than the liver. The strong upregulation of the CYP2A5 both at mRNA and enzyme activity levels, with a simultaneous decrease in the total CYP concentration suggest an unusual mode of regulation of CYP2A5 in response to cadmium exposure, amongst the CYP enzymes. The observed decrease in the mRNA but not in protein levels after maximal induction may suggest involvement of post-trancriptional mechanisms in the regulation. Upregulation of CYP2A5 by cadmium in the Nrf2 +/+ mice but not in the Nrf2−/− mice indicates a role for this transcription factor in the regulation. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Cadmium; Cytochrome P450 2A5; Coumarin 7-hydroxylase; Nrf2; Stress response gene

1. Introduction ∗

Corresponding author. Tel.: +61-7-3274-9060; fax: +61-7-3274-9003. E-mail address: [email protected] (A. Abu-Bakar).

The cytochrome P450 (CYP) monooxygenase system is critical for the metabolism of both endogenous and exogenous lipophilic substrates. The expression

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A. Abu-Bakar et al. / Toxicology Letters 148 (2004) 199–210

of many CYP genes is modulated by xenobiotics, and the regulation occurs at different levels ranging from transcriptional activation to protein stabilization (Porter and Coon, 1991). The transcriptional activation of CYP has been widely studied where a number of xenobiotics-activated receptors which act as specific transcription factors have been discovered (Waxman, 1999). Other than transcription, posttranscriptional control is important in the regulation of several CYPs (Silver and Krauter, 1990; Peng and Coon, 1998), however the molecular mechanism involved are poorly understood. To date, the post-transcriptional regulation of CYP2A5 expression has been more extensively studied than any other CYPs. CYP2A5 is upregulated post-transcriptionally through mRNA stabilization (Aida and Negishi, 1991) by the binding of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) to the 3 untranslated region (3 -UTR) (Geneste et al., 1996; Thulke-Gross et al., 1998; Glisovic et al., 2003). Such binding prevents the CYP2A5 mRNA from RNAse digestion and thus it prolongs the half-life of CYP2A5 mRNA. The activation of the CYP2A5 gene can also be achieved by transcriptional activation mechanisms (Aida and Negishi, 1991; Hahnemann et al., 1992). The CYP2A5 gene is reported to be induced by many structurally unrelated compounds, such as phenobarbital (Wood and Conney, 1974; Juvonen et al., 1985), pyrazole (Kojo et al., 1991), and metals (Kocer et al., 1990; Seubert et al., 2002). CYP2A5 is also induced following tissue damage caused by hepatitis B virus (Kirby et al., 1994). The mechanism(s) by which these xenochemical inducers and pathophysiological stressors, such as oxidant stress, upregulate hepatic CYP2A5 in mouse tissues is currently unknown. Cadmium (Cd) is one of the metals identified to be inducer of hepatic CYP2A5 catalytic activity (Urbenjapol et al., 2001) as well as of renal and hepatic CYP2A5 mRNA (Bartosiewicz et al., 2001). Cd has no known physiological function, but exposure to this metal is associated with renal dysfunction, early onset of diabetic renal complications, osteoporosis, hypertension, renal stone formation (Buchet et al., 1990; IARC, 1994; Alfen et al., 2000), cancer of various organs, such as kidney, lung (Kolonel, 1976, Sanderman, 1977), and prostate (Piscator, 1981). Induction of various hepatic chemical detoxication enzymes, such as NAD(P)H:quinone oxidoreductase

occurs rapidly following Cd treatment (Beyersmann and Hechtenberg, 1997). Similarly, induction of stress proteins including heme oxygenase-1 (HO-1), a certain heat shock protein, by Cd is well known (Alam et al., 1989; Applegate et al., 1991). In relation to this, Cd is reported to retard the degradation rate of the transcription factor nuclear erythroid-related factor 2 (Nrf2), which in turn leads to transcriptional activation of the HO-1 gene (Stewart et al., 2003). The objective of this study was to investigate the molecular mechanisms by which Cd affects the regulation of the CYP2A5 gene expression. More specifically, we determined the time course as well as dose response of the effects of CdCl2 treatment on CYP2A5 catalytic activity, mRNA, and protein levels, as well as total CYP content in kidney and liver of mice. Further, we attempted to demonstrate the involvement of Nrf2 in the activation of the CYP2A5 gene expression by Cd. Our interest in testing for Nrf2 involvement in Cd induction of the CYP2A5 gene stems from the observation that activation of the CYP2A5 gene is often found to be stress-related (Camus-Randon et al., 1996). In addition, Cd was shown to retard the degradation of Nrf2 protein (Stewart et al., 2003), which is known to be a general regulator of cell defence mechanism against oxidative stress. Here, we report the preliminary findings on the effects of CdCl2 treatment on CYP2A5 expression in Nrf2 knockout and wild-type mice.

2. Materials and methods 2.1. Materials and chemicals Cadmium chloride (purity 99%), coumarin, umbelliferone (7-hydroxycoumarin), NADPH, bovine serum albumin (BSA) and glycine were obtained from Sigma (Australia). Trizol Reagent was from Gibco (Australia). PVDF membrane, SeeBlue prestained protein molecular weight marker, Polyacrylamide/bis solution, goat anti-rabbit immunoglobulin conjugated with alkaline phosphatase, and alkaline phosphatase colour development reagents (BCIP/NBT) were obtained from Bio-Rad Laboratories Pty. Ltd. (Sydney, Australia). All other chemicals were of reagent grade available.


2.2. Animal treatment 2.2.1. Time-course and dose-response study Twenty-four 7–9 weeks old DBA/2J male mice were divided into six groups of four mice in each group. They were housed in filter-top polycarbonate cages containing wood chip bedding and maintained in a 12 h light/dark cycle with free access to standard mouse chow and tap water. The animals were treated i.p. with single dose of 16 ␮mol/kg body weight of CdCl2 dissolved in normal saline solution. The dose was chosen based on the results of our previous study, where CYP2A5 induction was observed (Urbenjapol et al., 2001). The animals in control group were given normal saline only. They were sacrificed at 4, 8, 12, 18 and 24 h after treatment by CO2 overdose and their livers and kidneys removed. For the dose-response study, the same number of animals were used and divided into six groups of four mice in each group. Each group were treated i.p. with 0, 5, 10, 15, 20 and 25 ␮mol CdCl2 /kg body weight, respectively for 18 h, at which time their livers and kidneys were remove. All the experimental procedures were approved by, and conducted in accordance with the animal experimentation guidelines of the Queensland Health Scientific Services and the University of Queensland Animal Ethics Committees. 2.2.2. Test for involvement of Nrf2 in Cd induction of Cyp2a5 Three wild-type (Nrf2 +/+) and 3 knock-out (Nrf2 −/−) ICR mice (male, 11–13 weeks old, 30 ± 10 g) (supplied by University of Tsukuba, Japan) were treated i.p. with single dose of 16 ␮mol/kg body weight of CdCl2 dissolved in normal saline solution. The animals were maintained as described above. The control mice received vehicle only (normal saline). They were sacrificed at 4 and 24 h after treatment and their livers and kidneys removed.

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(pH7.4). The pellet resulting from centrifugation of the homogenate (20 min, 4 ◦ C, 10,000 × g) was discarded and the supernatant filtered through MiraclothTM (Calbiochem) and centrifuged at 100,000 × g for 1 h. The pellet was collected and washed once in 100 mM potassium pyrophosphate buffer containing 1 mM EDTA and 20 ␮M BHT by resuspension and centrifugation at 100,000 × g for 1 h at 4◦ . The microsomal pellet was collected and resuspended in 10 mM Tris–acetate buffer containing 1 mM EDTA and 20% glycerol (pH 7.4). Protein concentrations of the microsomal samples (BSA as standard) were in the range of 22–56 ␮g/␮l (Lowry et al., 1951). The microsomal yield ranged from 2.5 to 10.0 mg/g wet liver weight. The samples were kept at −80◦ for later analysis. 2.4. Total cytochrome P-450 content determination Total microsomal CYP content was determined according to the carbon monoxide difference spectra method (Omura and Sato, 1964). Briefly, the suspension of microsomes was diluted to a final protein concentration of 1 ␮g/␮l in a final volume of 2.0 ml Sorensen’s buffer (containing 20 mM KH2 PO4 , 10 mM Na2 HPO4 , 1 mM EDTA, 20% glycerol, 0.5% sodium cholate, 0.4% Triton N-101, and 0.2% Emulgen 913), pH 4.7. The mixture was then transferred to 2 identical glass cuvettes (10 mm light path). The sample cuvette was treated with CO at the rate of 1 bubble per second. The microsomes in both the sample and reference cuvettes were reduced with sodium dithionite (2–3 mg). The concentration of CYP was obtained by the differential reading between the reduced protein, un-associated with CO (reference cuvette) and the reading obtained with the reduced protein-CO complex, using Cary 3E UV-Vis spectrophotometer (Varian Australia, Pty. Ltd). Wavelengths from 400 to 500 nm were used for the readings. 2.5. Coumarin 7-hydroxylase activity assay

2.3. Preparation of microsomes Liver and kidney microsomal fractions were prepared as described previously (Guengerich, 1994). Briefly, the tissue samples were homogenised using a motor-driven glass pestle and mortar in 0.1 M Tris–acetate buffer containing 0.1 M KCl, 1 mM EDTA, and 20 ␮M butylated hydroxytoluene (BHT)

The coumarin 7-hydroxylase (COH) activity was measured as previously described (Juvonen et al., 1985). Briefly, 300 ␮l of substrate buffer (160 mM potassium phosphate buffer (pH 7.4), 8 mM MgCl2 and 0.16 mM substrate coumarin) and 100 ␮l of microsomal sample (50 ␮g protein) were placed in a glass centrifuge tube. Reactions were started by the

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addition of 100 ␮l 7.5 mM NADPH and 20 min incubation at 37 ◦ C. Reactions were stopped by the addition of 500 ␮l of 6.5% trichloroacetic acid (TCA) and centrifuged at 2800 rpm for 5 min. Hydroxycoumarin formation was then determined spectrofluorometrically (Luminescence Spectrometer LS50B, Perkin Elmer, Ltd., UK) in 500 ␮l of supernatant plus 2 ml glycine/NaOH, pH 10.3 at excitation wavelength of 390 nm and emission wavelength of 440 nm. Catalytic activities were obtained by interpolation from a standard curve for hydroxycoumarin. A linear standard curve was achieved when the slit widths of the spectrofluorometer were set at 2.5 and 5.0 nm for excitation and emission, respectively. The photomultiplier tube (PMT) voltage was set at maximum (900 V) while the filter was fully opened. This condition was maintained throughout the experiments.

Finally, antibody detection was performed with the alkaline phosphatase colour development reagents (BCIP/NBT) system. Blots were than scanned with a scanner (HP Scanjet 3500 c) and the intensity of each bands were quantitated using the software NIH ImageJ (1.30), a public domain image-processing and analysis program for Windows (http://rsb.info.nih. gov/nih-image/).

2.6. Western blot

2.7.2. Northern blot analysis Total liver and kidney RNA (20 ␮g) was size-fractionated on a 1.2% agarose/formaldehyde gel and transferred to a HybondTM -N nylon membrane (Amersham Biosciences, Buckinghamshire, UK) The CYP2A5 cDNA (provided by Dr. Masahiko Negishi, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle, NC) was radiolabelled with [␣32 P]dCTP using the Megaprime labelling kit (Amersham Biosciences). Hybridization was performed with 1.7 × 107 cpm of radiolabeled probe at 65 ◦ C overnight in Church buffer (Church and Gilbert, 1984) modified to contain 0.25 M phosphate buffer, 7% SDS, and 1 mM EDTA. The membrane was washed 2 × 5 min at room temperature in a buffer containing 2 × SSC and 0.1% SDS and then 1 × 15 min at 65 ◦ C in a buffer containing 2 × SSC and 1% SDS. To assess equal loading of the samples, the mRNA level of the house keeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was measured using the GAPDH cDNA (CLONTECH, Palo Alto, CA) as a probe. For the densitometric analysis, scanning of the film was performed with a scanner (HP Scanjet 3500c), which was quantitated with the software NIH ImageJ (1.30).

CYP2A5 proteins in the liver and kidney microsomal suspensions were analysed by immunoblotting using liver and kidney microsomal fractions containing fixed amounts of protein. Liver and kidney microsomal proteins (10 and 20 ␮g protein, respectively) were separated by SDS-PAGE (4.5% acrylamide stacking gel, 9% acrylamide separating gel) in a Tris–glycine buffer system (Towbin et al., 1979). The prestained protein molecular weight markers were included in every SDS-PAGE, which was run along with six liver or kidney microsomal samples. The proteins on each gel were blotted onto a PVDF membrane for 1 h at constant voltage (100 V). Protein blots were blocked for 1 h at room temperature in blocking solution (20 mM Tris, 500 mM NaCl, 5% non-fat dry milk, pH 7.5). The blocking solution was removed and blots were rinsed 3 times in wash buffer (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5). Immunoblot analysis was performed by incubating with a primary monoclonal antimouse CYP2A5 antibody, kindly provided by Dr. M. Lang (Biomedical Centre, University of Uppsala, Sweden) (1:1000 dilution in 1% non-fat dry-milk-Tween-TBS) for 1 h at room temperature. The primary antibody solution was removed and blots were rinsed three times with wash buffer. Blots were incubated with the secondary antibody (alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin) for 45 min at room temperature.

2.7. CYP2A5 mRNA analysis 2.7.1. RNA isolation Total hepatic and renal RNA were prepared with TRIzol reagent according to manufacturer’s recommendation. RNA was resuspended in 50 ␮l DEPCtreated water and the concentration was determined optically at 260 and 280 nm after dilution in water.

2.7.3. RT-PCR Analysis of the CYP2A5 mRNA expression in the liver and kidney samples from Nrf2 +/+ and


Nrf2 −/− mice was conducted by RT-PCR. To obtain cDNA, reverse transcription (RT) was conducted at 42 ◦ C for 1 h in a total volume of 20 ␮l containing 3 ␮g of total RNA; 1.25 pmol oligo-(dT)12–18 ; 0.5 mM each of dATP, dGTP, dCTP, and dTTP; and 10 U M-MLV reverse transcriptase. PCR was carried out with a set of CYP2A5 and ␤-actin specific primers. The forward primer (sense) was 5 -ggcagctctatgagatgttc-3 , and the reverse primer (anti-sense) was 5 -ttatgaagtcctccaggccc-3 . Primers for mouse ␤-actin mRNA were used as an internal control. The forward primer (sense) was 5 -ATTGCTGACAGGATGCAGA-3 , and the reverse primer (antisense) was 5-GCTCAGGAGGAGCAATGATCTT-3 . PCR was performed in a total volume of 50 ␮l, using 1 ␮l of cDNA template containing 0.3 ␮M of the sense and anti-sense primers; 0.05 U of Taq DNA polymerase; 0.2 mM each of dATP, dGTP, dCTP, and dTTP; and 3 mM MgCl2 . Prior to amplification, PCR was ran at 95 ◦ C for 10 min to activate the Taq DNA polymerase. After amplification for 35 cycles (15 s at 95 ◦ C and 1 min at 58 ◦ C), the PCR products were analysed with 2% agarose gel stained with ethidium bromide. The mRNA expression levels were determined by scanning the agarose gel and by quantifying band intensities using the software NIH ImageJ (1.30). 2.8. Data analysis Data was analysed by the SPSS statistical package (version 11) for Windows. Parametric ANOVA with Dunnett’s test was used to determine statistical significance levels for differences in the total CYP among the groups. The non-parametric Kruskal–Wallis ANOVA with Mann–Whitney U-Wilcoxon Rank Sum W-test was used to determine significance levels for differences in the activities of COH among the groups.

3. Results 3.1. Effects of CdCl2 on total CYP content and coumarin 7-hydroxylase activity Treatment of mice with a single dose of CdCl2 (16 ␮mol/kg body weight) resulted in significant (P < 0.05) decrease in hepatic CYP content (22%) after

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12 h when compared to saline-treated control (Fig. 1a). Following this, CYP content continued to decline in a time-dependent manner with significant difference (>50% reduction) between treated and untreated mice from 18 h onwards. A significant reduction (55%) was seen in the kidney 8 h after treatment with a further reduction to more than 80% at 24 h (Fig. 1a). Interestingly, COH activity in the kidney and liver increased progressively with time after 4 h of treatment. Twofold at 8 h, threefold at 12 h, and sevenfold at 18 and 24 h increment were observed in the liver, whilst in the kidney fivefold increase in activity compared to control was observed at 8 h and further increased to eightfold at 24 h (Fig. 1a). Treatments with various doses of CdCl2 for 18 h showed progressive decrease in total CYP content in both the kidney and liver of treated animals compared to control (Fig. 1b). A significant 28% and 59% reduction (P < 0.05) was observed in the liver when treated with 15 and 20 ␮mol CdCl2 /kg body weight, respectively (Fig. 1b). A significant 80% reduction was seen in the kidney after treatment with 10 ␮mol CdCl2 /kg body weight. On the contrary, a significant 10-, 14-, 27-, 44-, and 50-fold increase in COH activity were seen in the liver at 5, 10, 15, 20 and 25 ␮mol CdCl2 /kg body weight, respectively (Fig. 1b). The fold induction, however is very much less in the kidney than in the liver (2-, 6-, 9-, and 15-fold at 10, 15, 20, and 25 ␮mol CdCl2 /kg body weight, respectively). 3.2. Effects of CdCl2 on CYP2A5 mRNA levels in liver and kidney samples from DBA/2J mice Figs. 2a and 3a show that the CYP2A5 mRNA in the kidney and liver of treated and control animals are detected by the radiolabeled probe. Densitometric analysis of the CYP2A5 mRNA band in the liver of animals treated with 16 ␮mol CdCl2 /kg body weight at various time points shows that its intensity is increased by a factor of 2, 11 and 13 at 4, 8, and 12 h, respectively. The band intensity decreased by a factor of 6 thereafter (Fig. 2b). In the kidney, the CYP2A5 mRNA band intensity increased by a factor of 5 at 4 h after treatment. The intensity remained the same at 8 h and started to decrease by a factor of 3 after 12 h (Fig. 2b). The basal CYP2A5 mRNA level is higher in the kidney than in the liver.

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Fig. 1. (a) Time course for changes in liver and kidney microsomal CYP content and COH activity of DBA/2J mice 4, 8, 12, 18, or 24 h after treatment with saline (control) or 16 ␮mol CdCl2 /kg body weight. Each data point represents the mean ± standard deviation of experiments performed with the liver and kidney of four individual animals. # Mean difference is significant from control group at P < 0.05 (one-way ANOVA with Dunnet test). ∗ Mean difference is significant from control group at P < 0.05 (Mann–Whitney U-test). (b) Dose response for changes in liver and kidney microsomal CYP content and COH activity of DBA/2J mice 4 after 18 h treatment with saline (control) or 5, 10, 15, 20, or 25 ␮mol CdCl2 /kg body weight. Each data point represents the mean ± standard deviation of experiments performed with the liver and kidney of four individual animals. # Mean difference is significant from control group at P < 0.05 (one-way ANOVA with Dunnet test). ∗ Mean difference is significant from control group at P < 0.05 (Mann–Whitney U-test).


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Fig. 2. (a) Effect of treatment with 16 ␮mol CdCl2 /kg body weight at different time points on CYP2A5 mRNA levels in the liver and kidney of DBA/2J mice. Northern blot analysis was performed using 20 ␮g total RNA isolated from treated and untreated mice (see Section 2) during the indicated time points. GAPDH mRNA levels are shown as controls for RNA loading. (b) Quantification of the CYP2A5 mRNA amounts in the liver and kidney of treated and untreated mice. The intensity of the band corresponding to the CYP2A5 mRNA shown. Each data point represents the mean ± standard deviation of experiments performed with the liver and kidney of four individual animals. (c) Western blot analysis of liver and kidney microsomes 4, 8, 12, 18, or 24 h after treatment with 16 ␮mol CdCl2 /kg body weight. Kidney samples were pooled from two animals in each group.

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Fig. 3. (a) Effect of 18 h of CdCl2 treatment at various doses on CYP2A5 mRNA levels in the liver and kidney of DBA/2J mice. Northern blot analysis was performed using 20 ␮g total RNA isolated from treated and untreated mice (see Section 2) during the indicated time points. GAPDH mRNA levels are shown as controls for RNA loading. (b) Quantification of the CYP2A5 mRNA amounts in the liver and kidney of treated and untreated mice. The intensity of the band corresponding to the CYP2A5 mRNA shown. Each data point represents the mean ± standard deviation of experiments performed with the liver and kidney of four individual animals. (c) Western blot analysis of liver and kidney microsomes 4, 8, 12, 18, or 24 h after treatment with 16 ␮mol CdCl2 /kg body weight. Kidney samples were pooled from two animals in each group.


Treatment with various doses of CdCl2 for 18 h showed progressive increase in the CYP2A5 mRNA band intensity in both the kidney and the liver. In the kidney, the band intensity increased by a factor of 2 after treatment with 15 ␮mol CdCl2 /kg body weight (Fig. 3b). Following that, the fold induction is constant at threefold compared to control. While in the liver the fold induction started to increase at a lower dose (sevenfold at 10 ␮mol). The intensity continues to increase by a factor of 12 and 21 thereafter and remains at 21-fold at 25 ␮mol CdCl2 /kg body weight (Fig. 3b). 3.3. Effect of CdCl2 on CYP2A5 protein abundance Western blot analysis was performed to ascertain whether the increase in CYP2A5 mRNA levels observed in the liver and kidney after CdCl2 treatment led to an increase in CYP2A5 protein. Increased CYP2A5

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protein levels were detected 8 h, 12 h, 18 h or 24 h after CdCl2 treatment, with the greatest increase in protein detected on 18th hour in the kidney and 24 h in the liver (Fig. 2c). Similarly, levels of CYP2A5 protein increased with dose 15, 20, or 25 ␮mol CdCl2 /kg body weight for 18 h. The highest increase in protein detected after treatment with 25 ␮mol CdCl2 /kg body weight in both the liver and kidney (Fig. 3c). 3.4. Effect of CdCl2 on CYP2A5 mRNA levels in liver and kidney of Nrf2 (+/+) and Nrf2 (−/−) mice Fig. 4a presents expression profile in 5 mice which shows CYP2A5 mRNA were detectable in liver and kidney of treated and untreated Nrf2+/+ and Nrf2−/− mice. Analysis with densitometry shows higher level of expression in liver of treated wild-type mice than untreated animal (Fig. 4b). There was no change in level of expression in liver of treated and

Fig. 4. (a) Expression profile of CYP2A5 mRNA in the liver and kidney of ICR mice. Wild-type (Nrf2 +/+) and knock-out (Nrf2 −/−) ICR male mice were treated (i.p.) with 16 ␮mol CdCl2 /kg body weight for 4 and 24 h (see Section 2). Knock-out mouse treated for 24 h died at 18 h. (b) Densitometric analysis of (a) using the software NIH ImageJ (1.30). ∗ Values are given as arbitrary densitometric units.

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untreated knockout mice. The same pattern was also observed in the kidneys (Fig. 4b).

4. Discussion The present study demonstrates that CdCl2 treatment upregulates the expression of CYP2A5 while it reduces the total CYP content in both the kidney and liver of mice. The effects are time and dose dependent. Our observation that total CYP content decreases with time following CdCl2 treatment is in tandem with the fact that Cd is a potent inducer of haem oxygenase-1 (HO-1) (Eaton et al., 1980; Alam et al., 1989; Applegate et al., 1991), which is an enzyme responsible for the catabolism of haem. HO-1 can contribute directly to the decrease of CYP and CYP-catalysed activity by increased degradation of “free” haem, making it unavailable for incorporation into CYP protein (Karuzina and Archakov, 1994). Indeed, earlier in vivo study, where rats were treated (s.c.) with 7 ␮mol CdCl2 for 16 h showed 100% increase in hepatic HO-1 activity and 60% reduction in total hepatic CYP content (Maines, 1984). The opposing observation that CYP content decreases while COH activity increases with time corresponds to the earlier findings that upregulation of CYP2A5 by various xenobiotics may occur when total CYP content decreases and HO-1 activity increases, for example following CCl4 treatment (Pellinen et al., 1993) or As3+ treatment in mouse kidney (Seubert et al., 2002). These observations suggest a compensatory feedback mechanisms to maintain the CYP2A5 activity when intracellular haem levels are reduced. Interestingly, this seems to be a rather specific phenomenon for CYP2A5 amongst the various drug metabolising CYP-enzymes, which could indicate a yet unknown biological role for the CYP2A5. The mechanisms for such feedback are currently unknown. It is noteworthy that, CdCl2 treatment in our present study showed a progressive increase in microsomal COH activity (Fig. 1) but not 7-ethoxyresorufin o-deethylase (EROD) activity (results not shown). The COH activity is exclusively attributable to CYP2A5, while EROD activity is mainly attributable to CYP1A1/1A2. Thus, these catalytic activities are useful markers for differential effects of

acute Cd administration on various CYP forms. Because we do not detect any changes in EROD activity in the liver of treated mice, our findings demonstrate a selective increase of catalytic activity of a CYP2A5 after CdCl2 treatment. The mechanism of upregulation of hepatic and kidney CYP2A5 by cadmium is not apparent in this study. However, the observation that the CYP2A5 mRNA levels (both in the kidney and liver) progressively decreased 12 h after treatment (Fig. 2), while the protein levels progressively increased with time (Fig. 2), suggests that the upregulation of CYP2A5 at that time was mediated, at least in part, at the post-transcriptional level. In addition, the observation of no change in the expression of CYP2A5 mRNA in the liver and kidney of treated knock-out (Nrf−/−) mice, in contrast to the changes seen in the wild-type (Nrf2+/+) mice suggests that Cd induction of CYP2A5 requires the transcription factor Nrf2. Of relevance to Nrf2-dependent effect of Cd on CYP2A5 expression is a more recent study showing that Nrf2 is rapidly degraded via the ubiquitinproteasome pathway and that Cd retards the rate of Nrf2 degradation leading to ho-1 gene transcription activation (Stewart et al., 2003). Another study attempted to characterise the regulatory factors involved in the post-transcriptional regulation of CYP2A5 gene, Tilloy-Ellul et al. (1999) found that the 71 nt long putative hairpin-loop of the CYP2A5 mRNA 3 -UTR formed a 37/39 kDa and a 70/72 kDa complex with protein extracts. The 37/39 kDa was later found to be hnRNP A1 and was shown to regulate CYP2A5 gene expression both at post-transcriptional and transcriptional levels (Raffalli-Mathieu et al., 2002; Glisovic et al., 2003). The composition of the 70/72 kDa complex was not pursued. Interestingly, however, Nrf2, has been reported to be a 72 kDa protein (Alam et al., 1999). Taken together, these observations, the Nrf2 dependent nature of cadmium induction of the CYP2A5 revealed for the first time in our present study, and with the established role of hnRNP A1 in CYP2A5 regulation (Geneste et al., 1996; Raffalli-Mathieu et al., 2002), we postulate that Nrf2 could be involved in the expression of the CYP2A5 gene possibly by interacting with hnRNP A1 at DNA or mRNA levels. It is also possible that the two proteins interact indirectly via a third transacting factor. These aspects of molecular mechanisms by which Cd


affects the regulation of CYP2A5 gene expression is under investigation in our laboratory. Acknowledgements We thank Dr. Masayuki Yamamoto for the generous gift of the Nrf2 knock-out ICR mice. We are grateful to Dr. Tetsuya Saito and Professor Tetsuya Kamataki, Laboratory of Drug Metabolism, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan for technical contributions. A.A.B is a recipient of the University of Queensland International Postgraduate Research Scholarship (UQIPRS) and the International Postgraduate Research Scholarship (IPRS). Queensland Health provides funding for the National Research Centre for Environmental Toxicology (EnTox).

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