O CYP1B1 CYP1A1 +DNA meh HO OH H NO 2. N OR NATs NQO1 N OH. SULTs

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1 CYP CYP CYP +DN meh H H H H enzo[a]pyrene (ap) ap-7,8-dihydrodiol ap-7,8-dihydrodiol-9,0-epoxide (PDE) H H N N H N R NTs NQ SULTs +DN DN adducts DN adducts -Nitrobenzanthrone (-N) N-Hydroxy--aminobenzanthrone (N-H--) N-cetoxy- or N-Sulfooxy--aminobenzanthrone Supporting Figure Main metabolic pathways in the bioactivation and DN adduct formation in vivo of ap () and -N (). R = C()CH ; R = S H. See text for details. CYP, cytochrome P50; meh, microsomal epoxide hydrolase; NQ, ND(P)H:quinone oxidoreductase; NT, N- cetyltransferase; SULT, sulfotransferase.

2 standards Supporting Figure Genotyping example for the mouse Trp5 allele in liver DN isolated from, and mice exposed to ap. DN was subjected to allele-specific primers and resolved on a % agarose gel containing ethidium bromide as described (Jacks et al. 99).

3 5 liver lung kidney liver colon small intestine bladder lung glandular stomach fore stomach spleen kidney Supporting Figure utoradiographic profiles of DN adducts, measured by P-postlabelling, in various tissues of mice exposed to ap () or PDE (). The adduct profiles shown are representative for the same organs in and mice. Solvent conditions for the separation of ap-derived DN adducts were as follows: D,.0 M sodium phosphate, ph 6.0; D,.5 M lithium-formate, 8.5 M urea, ph.5; D, 0.8 M lithium chloride, 0.5 M Tris, 8.5 M urea, ph 8.0. The origins, at the bottom left-hand corners, were cut off before exposure. Spot, 0-(deoxyguanosin-N -yl)-7,8,9-trihydroxy-7,8,9,0-tetrahydro-ap (dg- N -PDE); Spot, probable guanine adduct derived from reaction with 9-hydroxy-aP-,5- epoxide; Spot, uncharacterised ap-derived DN adducts. For the pathways of ap-dn adduct formation see Supporting Figure 6.

4 6 liver lung kidney colon small intestine bladder glandular stomach fore stomach spleen Supporting Figure utoradiographic profiles of DN adducts, measured by P-postlabelling, in various tissues of mice exposed to -N. The adduct profiles shown are representative for the same organs in and mice. -N-derived DN adducts were separated using the following solvent conditions: D,.0 M sodium phosphate, ph 6.0; D,.0 M lithium-formate, 7.0 M urea, ph.5; D, 0.8 M lithium chloride, 0.5 M Tris, 8.5 M urea, ph 8.0. The origins, at the bottom left-hand corners, were cut off before exposure. Spot, -( - deoxyadenosine-n 6 -yl)--aminobenzanthrone (d-n 6 --); Spot, as-yet unidentified adenine adduct derived from nitroreduction; Spot, N-( -deoxyguanosine-n -yl)-- aminobenzanthrone (dg-n --); Spot, N-( -deoxyguanosin-8-yl)--aminobenzanthrone (dg-c8-n--).

5 7 RL per 0 8 nucleotides dg-n -PDE Supporting Figure 5 ap-dn adducts, measured by P-postlabelling, formed ex vivo by hepatic microsomes isolated from control (untreated), and mice. Values are the mean ± range (n = ); duplicate incubations and each sample was determined by two independent post-labelled analyses. Statistical analysis was performed by one-way NV followed by Tukey post-hoc test; no significant differences were observed. Inserts: utoradiographic profiles of DN adducts formed in hepatic microsomes isolated from mice; the origins, at the bottom left-hand corners, were cut off before exposure.

6 8 CYP 9 0 CYP (+)-ap-9,0-epoxide ap (+)-ap-7,8-epoxide meh H H CYP 9-Hydroxy-aP H (-)-trans-ap-7,8-dihydrodiol CYP H +DN dduct 9-Hydroxy-(+)-aP-,5-epoxide ap-treated lung dduct (dg-n -PDE) Hepatic microsomes ex vivo +DN H H (+)-anti-ap-7,8-dihydrodiol- 9,0-epoxide (PDE) (dg-n -PDE) (dg-n -PDE) Supporting Figure 6 Pathways of biotransformation and DN adduct formation of ap catalysed by CYP and microsomal epoxide hydrolase (meh) [adapted from (Stiborova et al. 0)]. The typical three-step activation process with oxidation by CYP followed by hydrolysis by meh leads to the ultimately reactive species PDE which leads to the generation of the dg-n - PDE adduct. The two-step activation process by CYP is leading to the formation of the ultimately reactive species, 9-hydroxy-aP-,5-epoxide, that can react with deoxyguanosine in DN (adduct ; structure unknown). Insert: utoradiographic profiles of ap-dn adducts, measured by P-postlabelling, formed in the lungs of ap-treated mice (see also Supporting Figure ) or ex vivo in hepatic microsomes isolated from appretreated mice (see also Figure ); the origin, at the bottom left-hand corners, was cut off before exposure.

7 9 C D E mu [5 nm] mu [5 nm] mu [5 nm] mu [5 nm] mu [5 nm] 50.0 mu WVL:5 nm -.0 min mu WVL:5 nm -.0 min mu WVL:5 nm -.0 min mu WVL:5 nm -.0 min mu M Mx M M M M5 M6 M7 ap WVL:5 nm -.0 min Retention time Hepatic microsomal incubation; aptreated mice; with NDPH and with addition of ap Hepatic microsomal incubation; ap-treated mice; without NDPH but with addition of ap Hepatic microsomal incubation; ap-treated mice; with NDPH but without addition of ap Hepatic microsomal incubation; ap-treated mice; with NDPH but without addition of ap Hepatic microsomal incubation; ap-treated mice; with NDPH but without addition of ap Supporting Figure 7 () Representative HPLC chromatogram of the ap metabolites generated hepatic microsomal incubations of ap-pretreated with NDPH and ap. () Representative HPLC chromatogram of the ap metabolites generated hepatic microsomal incubations of ap-pretreated with ap but without NDPH. Representative HPLC chromatogram of the ap metabolites generated hepatic microsomal incubations of ap-pretreated (C), (D) and mice (E) without the addition of ap.

8 0 H 9 H 0 5 H H 8 7 M = ap-9,0-dihydrodiol M = ap-,5-dihydrodiol H H M = ap-7,8-dihydrodiol H 9 H 6 6 M = ap-,6-dione M5 = ap-,6-dione M6 = ap-9-ol M7 = ap--ol Supporting Figure 8 Structures of ap metabolites analysed by HPLC analysis.

9 ap-9,0-dihydrodiol ap-,5-dihydrodiol ap-7,8-dihydrodiol ap-,6-dione ap-,6-dione ap-9-ol ap--ol Mx C ap-9,0-dihydrodiol ap-,5-dihydrodiol ap-7,8-dihydrodiol ap-,6-dione ap-,6-dione ap-9-ol ap--ol Mx Supporting Figure 9 Formation of ap metabolites by hepatic microsomes isolated from control (untreated) (), () and mice (C). Relative peak areas of ap metabolites were measured by HPLC analysis at 5 nm. Values are the mean ± SD (n = ). Statistical analysis was performed by one-way NV followed by Tukey post-hoc test; no significant differences were observed. Structures of the ap metabolites detected by HPLC are shown in Supplementary Figure 8. Mx, an unknown ap metabolite.

10 liver - + p5 Gapdh ap Supporting Figure 0 Western blot analysis of p5 in the livers of mice exposed to ap. Representative images of the Western blotting are shown; at least duplicate analysis was performed from independent experiments. Gapdh protein expression was used as loading control.

11 C E G relative ERD activity relative NQ activity relative NQ activity Supporting Figure * * liver (ap) liver (-N) 5 relative ERD activity D F H relative NQ activity lung (untreated) 0 relative ERD activity relative NQ activity 0 lung (-N) 5 ERD activity (-D) in hepatic ( and C) and pulmonary microsomes ( and D) isolated from, and mice. Nqo enzyme activity (E-H) was determined in hepatic (E and G) and pulmonary cytosols (F and H). Values are the mean ± SD (n = ). Statistical analysis was performed by one-way NV followed by Tukey posthoc test (*p<0.05; different from mice).

12 relative repair capacity [%] * Supporting Figure relative repair capacity [%] NER capacity in liver () and kidney () of, and mice as assessed by the comet assay. Values are the mean ± SD (n = ). Statistical analysis was performed by one-way NV followed by Tukey post-hoc test (*p<0.05; different from mice).

13 5 mu [5 nm] 50.0 mu M M MM5 M6 M7 ap WVL:5 nm -.0 min Retention time ** ** ** ND ap-7,8-dihydrodiol ap-,6-dione ap-9,0-dihydrodiol ap-,5-dihydrodiol ap-,6-dione ap-9-ol ap--ol Mx ND Supporting Figure () Representative HPLC chromatogram of the ap metabolites found in urine of mice exposed to ap. () ap metabolites in urine of ap-treated and mice. Relative peak areas of ap metabolites were measured by HPLC analysis at 5 nm. Values are the mean ± SD (n = ). Statistical analysis was performed by t-test analysis (**p<0.0, ***p<0.005; different from mice). Mx, an unknown ap metabolite. ND, not detected.