XENOBIOTICA,
1991, VOL. 21,
NO.
7, 971-977
The metabolism of 4-aminobiphenyl in rat. IV. Ferrihaemoglobin formation by 4-aminobiphenyl metabolites S. KARRETH and W. LENKT Walther Straub-Institut fur Pharmakologie und Toxikologie der LM-Universitat Miinchen, D-8000 Munchen 2, NuRbaumstr. 26, Germany
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
Received 26 March 1990; accepted 1 November 1990
1. Rats dosed with nitrosobenzene (56pmol/kg), 4-chloronitrosobenzene (53 pmol/kg), 3,4-dichloronitrosobenzene (53pmol/kg), 4-ethoxynitrosobenzene (86pmol/kg), 4nitrosobiphenyl(nitroso-BP, 55 pmollkg) or 2-nitrosofluorene (256 pmol/kg) had maximal ferrihaemoglobin (HbFe") concn of 69,68, 69,67, 55 and 42% after 15,25,48, 35, 80 and 115 min, respectively, indicating differences in solubility of the nitrosoarenes in body fluids. 2. Nitroso-BP and 3-hydroxy-4-aminobiphenyl (3-hydroxy-ABP) catalytically oxidized HbFe2' in bovine erythrocytes in vitro; nitroso-BP was three times as active as 3-hydroxy-ABP. 3',4'-Dihydroxy-4-aminobiphenyl (3',4'dihydroxy-ABP) showed only low catalytic activity, and seven other ABP metabolites exhibited only marginal activity. 3. Nitroso-BP was inactive in solutions of purified human Hb, but 3-hydroxy-ABP catalytically oxidized HbFe", indicating that nitrosoarenes oxidize HbFe'' in erythrocytes in vitro and in oivo by a mechanism different from that of o-aminophenols. T h e second-order rate constant for HbFe*+ oxidation by 3-hydroxy-ABP at 37°C was k2=19.1f1~31/molper s.
Introduction If ferrihaemoglobin (HbFe3+) is formed in vivo after administration of an arylamine which does not form HbFe3+ in vitro, HbFe3+ formation is due to active metabolites produced by N- and/or ring-hydroxylation in the liver. T h e role which arylhydroxylamines and aminophenols play during HbFe3+ formation in vivo merits investigation because HbFe3'-forming activity is a further aspect of drug metabolism which has consequences for the acute or chronic toxicity of the arylamine. 4-Aminobiphenyl (ABP), for example, the strong HbFe3+-forming activity of which was first observed by Klingenberg (1891) in dog, is metabolized by rat liver enzymes in vitro to N-hydroxy-4-aminobiphenyl(N-hydroxy-ABP), 3-hydroxy-4aminobiphenyl(3-hydroxy-ABP),2'-hydroxy-4-aminobiphenyl(2'-hydroxy-ABP) and 4'-hydroxy-4-aminobiphenyl(4-hydroxy-ABP) (McMahon et al. 1980). After injection of ABP into dogs, rabbits and cats, N-hydroxy-ABP was found in the blood (von Jagow et al. 1966, Uehleke and Nestel 1967), as a sensitive assay (Herr and Kiese 1959) was available. Because no such method was available for the phenolic metabolites, these were not determined, and therefore it remained unknown whether or not they play any role during ABP-induced HbFe3+ formation in vivo. T h e HbFe3+-forming activity of N-hydroxy-ABP, in comparison with other arylhydroxylamines, was studied in rat (Lenk and Sterzll982) and in vitro (Heilmair et al. 1987, Lenk and Sterzl 1987); we now report studies of the HbFe3+-forming activity of 4-nitrosobiphenyl (nitroso-BP) in comparison with other relevant nitrosoarenes in vivo and of 3-hydroxy-ABP and other metabolites of ABP in comparison with N-hydroxy-ABP in vitro.
7 Author to whom correspondence should be addressed. 0049-8254/91 $3.00 0 1991 Taylor & Francis Ltd
972
S. Karreth and W. Lenk
Materials and methods
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
Chemicals Nitrosoarenes. Commercial nitrosobenzene was recryst. from ethanol prior to use. 4-Nitrosobiphenyl was obtained by oxidation of N-hydroxy-4-aminobiphenylwith periodate as described (Karreth and Lenk 1991 a); U.V.absorption in methanol showed A,,, = 341 and 231 nm. 2-Nitrosofluorene was prepared from 2-nitrofluorene according to von Jagow et al. (1966); the crude product was purified by column chromatography and crystn from n-hexane and showed A,, = 357, 260 and 240 nm in methanol. Other nitrosoarenes were obtained from the corresponding arylhydroxylamines by oxidation with FeCI,, steam distillation of the precipitate and recrystn from small volumes of n-heptane or ethanol at -27°C; U . V . absorption in methanol, 4-chloronitrosobenzene, A,,= 313, 288 and 223 nm; 3,4-dichloronitrosobenzene, A,, = 31 1, 289 and 231 nm; 4-ethoxynitrosobenzene, A,, = 341 and 239 nm. Derivatives of 4-aminobiphenyl. Preparation and properties of 3-hydroxy-, 2'-hydroxy-, 4'-hydroxy- and 3',4-dihydroxy-4-aminobiphenyl,3-hydroxy-, 2'-hydroxy-, 4-hydroxy- and 3',4-dihydroxy-4acetylaminobiphenyl, 3',4'-dihydroxy-4-nitrobiphenyl and 4-(4-aminophenyl)-l,2-henzoquinoneare described elsewhere (Karreth and Lenk 1991b). Biological material Bovine blood was obtained from the slaughter-house and mixed with heparin to prevent clotting. Purified human haemoglobin A was prepared from outdated human blood concentrate according to Heilmair et al. (1987). Animals Female Sprague-Dawley rats of 20&300g body wt were housed in Acme stainless steel metabolism cages, and fed Alma Standard diet for mice and rats (0801-H 10031, D-8960 (Kempten), with water ad libitum. Methods Dosing rats with nitrosoarenes. Nitrosoarenes were finely ground, suspended in 0.25% agar soln (prepared from 0.9% saline) and injected i.p. in rats. At the times indicated, 3 drops of blood were withdrawn from the tail vein for the determination of HhFe3' and total IIbFe''. Bovine blood was separated into plasma and packed erythrocytes by centrifugation at +4"C. T h e packed erythrocytes were washed three times with 0.9% saline and resuspended in Krebs-Ringer phosphate buffer pH 7.4 without additives. Addition ojsubstrotes. After shaking suspensions of red cells or solns of purified haemoglobin at 37°C in a water-bath for 5 min, methanolic solns of the substances were added and aliquots of 0.1 ml removed at times indicated. HbFe3' concentration and total HbFe2' in rat blood, suspensions of bovine erythrocytes or s o h of purified haemoglobin were determined according to Evelyn and Malloy (1938) and modified by Kiese (1974). Aliquots of 0.1 ml blood (in duplicate) or red cell suspension were haemolysed with lOml ice-cold water, 1 ml 0.2 M Sorensen buffer pH 6.6 added, centrifuged and the light absorption of the clear supernatant was measured at 550nm.
Results Ferrihaemoglobin formation in rats by six nitrosoarenes Following i.p. injection of nitrosobenzene (NOB), 4-chloronitrosobenzene (4-CINOB), 3,4-dichloronitrosobenzene (3,4-CI2NOB), 4-ethoxynitrosobenzene (4-ENOB), 4-nitrosobiphenyl (4-NOBP) and 2-nitrosofluorene (2-NOF), HbFe3 concns in rat blood were monitored for 5 h (figure 1). In the first phase of the reaction with HbFe", a time-dependent increase in HbFe3+ concn occurred which lasted as long as HbFe3+ formation exceeded enzymic HbFe3+ reduction. However, when the reactive nitroso compounds were inactivated by metabolism, HbFe3+ formation ceased and HbFe3+ concn passed its maximum. Thereafter HbFe3+ reduction decreased the concn of HbFe3+ in the second phase of the reaction. As can be seen from figure 1, the nitrosoarenes differed in the initial velocity of HbFe3+ formation, both in the time and magnitude of the maximal HbFe3+ concn, and in the subsequent decline of the HbFe3+ concn. +
HbFe3+ formation by 4-aminobiphenyl metabolites
973
90 -
0
z
70
50
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
30
10 1
2
3
4
5
Time a f t e r d o s i n g ( h ) Figure 1.
Kinetics of ferrihaemoglobinformation in oiwo after i.p. injection of various nitrosoarenes to female rats.
Rats were dosed with nitrosobenzene (56pmol/kg, -A-),
4-chloronitrosobenzene (53 pmol/kg,
-0-), 3,4-dichloronitrosobenzene(53 pmollkg, -0-),4-ethoxynitrosobenzene (86 pmol/kg,
-Ap), 4-nitrosobiphenyl (54.6pmol/kg, -0-) and 2-nitrosofluorene (256pmol/kg, -B-). For i.p. injection, the compounds were finely ground, suspended in 0.25%agar s o h , and injected.
Symbols indicate mean values from three animals each.
Due to differences in solubility in body fluids, such as lymph and blood, the four monocyclic nitroso compounds were active from the first minute, whereas nitrosoBP and 2-NOF became active after a lag phase of 5 min. Differences in the initial velocity of HbFe3+ formation, of 6.7%/min for NOB, 55%/min for 4-ClNOB, 3.3%/min for 3,4-C12NOB, 3.0%/min for 4-ENOB, 1.4%/min for nitroso-BP and 0.6%/min for 2-NOF showed, however, that differences in solubility exist also among the monocyclic nitroso compounds, although these were not as marked as between the monocyclic and polycyclic analogues. Maximal HbFe3 concns of 69% produced by 56pmol/kg NOB were determined after 15 min, 67% HbFe3+ by 53 pmol/kg 4-ClNOB after 25 min, 69% HbFe3+ by 53 pmol/kg, 3,4-C12NOB after 45min, 67% HbFe3+ by 86pmollkg 4-ENOB after 35min, 55% HbFe3+ by 546 pmol/kg nitroso-BP after 75 min, and 42% HbFe3+ only by 256pmol/kg 2NOF after 110 min. Thereafter HbFe3+ concn declined most rapidly at a rate of 24%/h after dosing rats with NOB, followed by 4-ENOB at a rate of 18%/h,while the others produced a decline at a rate of 14%/h. This is an indication that metabolism and HbFe3+ reduction are related to each other, since both NOB and 4-ENOB are rapidly metabolized in rats. HbFe3' formation by 2-NOF was sluggish and inefficient, because a five-fold higher dose produced a maximum of 42% HbFe3+ only. T h e reason for the ineffective and protracted HbFe3+ formation may be found in the low solubility of 2-NOF in lymph and blood and in its incomplete mobilization from the peritoneal cavity, as residual 2-NOF was found remaining in the peritoneal cavity some days later. +
S . Karreth and W . Lenk
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
974
Ferrihaemoglobin formation in suspensions of bovine erythrocytes Suspensions of washed bovine erythrocytes (HbFe2+concn: 5 mM) incubated at 37°C with 110, 50 or 1 O p nitroso-BP, ~ oxidized 26, 63 and 150equiv. of H b F e 2 + , respectively, in 1 h. After 2 h, 33, 71 and 215 equiv. of H b F e 2 + , respectively, were oxidized (figure 2). This is an indication that nitroso-BP oxidized H b F e 2 + catalytically and that the catalytic activity increased with an increasing ratio of nitrosoarene : HbFe2 +. This finding is in agreement with the mechanism of haemoglobin oxidation in erythrocytes in vivo and in vitro by arylhydroxylamines (Kiese et al. 1950, Heilmair et al. 1991). In suspensions of washed bovine erythrocytes (HbFe2+ concn: 5 mM) incubated at 37°C with 1OmM 3-hydroxy-ABP, H b F e 3 + was rapidly formed, and within 1 h H b F e 2 +was completely oxidized (figure 3). With 1-23mM 3-hydroxy-ABP the rate of HbFe3+ formation was the same, but the reaction was finished after 45 min, when 91% HbFe3+ was formed. Similarly 93 p M 3-hydroxy-ABP oxidized 8.6 equiv. of HbFe’ in 15 min, 28 equiv. after 1 h and 39 equiv. after 2 h; 50 p~ 3-hydroxy-ABP oxidized 4.7equiv. of HbFe” after 15 min, 21 equiv. after 1 h, and 49 equiv. after 2 h, indicating that the catalytic activity of 50 PM 3-hydroxy-ABP was only one-third of the catalytic activity of nitroso-BP. On incubation at 37°C of suspensions of washed bovine erythrocytes (HbFe” concn: 5 mM) with 10mM 3‘,4’-dihydroxy-ABP, 62% HbFe3+ was formed after 1 h, 85% after 2 h and 100%after 3 h, i.e. each 3’,4’-dihydroxy-ABP had oxidized 0.3,0.4 and 0.5 equiv. of H b F e 2 + ,respectively; with 5 mM 3’,4’-dihydroxy-ABP, each mmol oxidized 0.6, 0 8 and 0.95 equiv. of H b F e 2 + , and with 1 m M 3’,4’-dihydroxy-ABP, each mmol oxidized 1.3 equiv. of HbFe2+ in 1 h, 2.0 equiv. in 2 h and 2.5 equiv. in 3 h, indicating low catalytic activity of 3’,4’-dihydroxy-ABP at a molar ratio of 1 : 5 . 3-Hydroxy-AABP, 2’-hydroxy-ABP, 2’-hydroxy-AABP, 4’-hydroxy-ABP, 4’hydroxy-AABP, 3’,4‘-dihydroxy-AABP and 4-(4-aminophenyl)-l,2-benzoquinone, which were also examined for their HbFe3+-forming activity in suspensions of bovine erythrocytes, exhibited only marginal activity and lacked catalytic activity. +
Ferrihaemoglobin formation in solutions of purified haemoglobin On incubation of purified haemoglobin (HbFe2+ concn: 5.1 mM) at 37°C with 6 7 p nitroso-BP ~ for 3.5 h, 8.9% HbFe3+ was formed, indicating that not more HbFe3+ was formed than was produced by autoxidation of HbO,. This proves the inability of the nitrosoarene to oxidize H b F e 2 + , unless it is activated by enzymic reduction to the arylhydroxylamine. On incubation of purified haemoglobin (HbFe” concn: 5.1 mM) at 37°C with 6 7 p 3-hydroxy-ABP, ~ 41% HbFe3+ was formed in 1.5 h, i.e. each 3-hydroxy-ABP has oxidized 24 equiv. of HbFe*+. In contrast to nitroso-BP, the o-aminophenol displayed catalytic activity, which shows that the mechanism of HbFe” oxidation by o-aminophenols differs from that of nitrosoarenes. T h e order of the reaction of 3-hydroxy-ABP with H b F e 2 + was determined by measuring initial velocities of HbFe3+ formation at a constant concn of HbFe” (155 p ~ with ) varying concns of 3-hydroxy-ABP (5-100 PM), and constant concn of 3-hydroxy-ABP ( 1 0 0 ~ with ~ ) varying concns of H b F e 2 + ( 1 3 0 p ~ - lmM). O n plotting initial velocities of HbFe3+ formation versus concn of either H b F e 2 + or 3hydroxy-ABP in a double-logarithmic scale, two parallel straight lines with a slope of 1 were obtained, indicating that haemoglobin oxidation by 3-hydroxy-ABP was first order with respect to either reactant, and second order with respect to HbFe3+
HbFe3 formation by 4-aminobiphenyl metabolites
975
+
0
5!
roo
X L
\
50
0
10pM
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
m Time of incubation (h) Figure 2.
Kinetics of ferrihaemoglobin formation in erythrocytes in witro by 4-nitrosobiphenyl.
Suspensions of washed bovine erythrocytes (HbFe” concn: 5 mM) in Krebs-Ringer phosphate buffer, pH 7.4, were incubated at 37°C with various concns of nitroso-BP and HbFe3+ determined at times indicated. Symbols are from single experiments.
0
z
100
X
60
20 2
3
5
L
Time of incubation(h) Figure 3.
Kinetics of ferrihaemoglobin formation in erythrocytes in witro by 3-hydroxy-4aminobiphenyl.
Suspensions of washed bovine erythrocytes (HbFe” concn: 5 mM) in Krebs-Ringer phosphate buffer, pH7.4, were incubated at 37°C with various concns of 3-hydroxy-ABP and HbFe” determined at times indicated. Symbols are from single experiments.
formation. Based on these results from table 1, the second-order rate constant was calculated to be k2 = 19.1& 1.3 I/mol per s.
Discussion The results presented here show that nitroso-BP catalytically oxidized HbFe2+ in erythrocytes, but was inactive in solutions of purified haemoglobin. In contrast, 3-hydroxy-ABP catalytically oxidized HbFe2+both in erythrocytes and in solutions of haemoglobin, indicating differences in the mechanism of HbFe’ oxidation between these two classes of compounds. Kiese et al. (1950) recognized that nitrosoarenes require enzymic reduction to the active arylhydroxylamine for the oxidation of many equiv. of HbFe” in erythrocytes in vivo and in vitro. Therefore in solutions of purified haemoglobin nitrosoBP cannot be reduced to the active arylhydroxylamine. +
S. Karreth and W . Lenk
976
Table 1. The rate coefficient of haemoglobin oxidation by 3-hydroxy-4-aminobiphenyldetermined from initial velocities. 3-Hydroxy-4-aminobiphenyl
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
(mM)
Initial HhFe” (mM)
Formation of HhFe3’ (mM/s)
k2
(limo1 per
s)
0.1 0.05 0.0 1 0.005
0.155 0.155 0155 0.1 55
0.24 x 10-37 0 1 4 10-3 ~ 0.03 x 10-3 002 x 10-3
15.5 18.1 25.8 25.8
0.1 0.1 0.1 0.1 0.1
1.0 0.45 0.291 0.223
1.87 x 10-31 077 x 0.51 x 10-3 0.40 x 10-3 0 . 2 0 ~10-3
18.7 17.1 17.5 17.9 15.4 19.1 5 1.3 (SEM)
0.13
t Autoxidation of Hb: 0,155mM HhFe”: 0 0 8 6 p ~ / m i n . $Autoxidation of Hb: 1 mM HbFe”: @ 8 6 p ~ / m i n . 0 1 3 0 m ~HbFeZt: 0.152p~lrnin. It was demonstrated that aminophenols catalytically oxidize HbFe’ in red cells in nitro by Eyer et al. (1974) with 4-dimethylaminophenol (DMAP). At a molar ratio of DMAP : HbFe2+of 1 :42, each mol of DMAP oxidized an average of 60 equiv. of HbFe”, and its catalytic activity depended on the molar ratio of DMAP : HbFe”. Experiments with l4C-labe1led DMAP have shown that all activity was bound to the globin moieties. T h e mechanism of catalytic oxidation of HbFeZ+by DMAP was elucidated by Eyer and Lengfelder (1984). DMAP was oxidized by the activated oxygen of HbO, to the phenoxy radical via one-electron transfer, and the phenoxy radical in turn oxidizes HbFe2+ and is reduced back to DMAP. But the phenoxy radical also disproportionates into the quinoneimine and DMAP, and the quinoneimine is bound to the SH-groups of globin (and G S H in erythrocytes) to give thioethers. Any 3-hydroxy-ABP, which was produced in the liver of rats after dosing with ABP, therefore, participated with N-hydroxy-ABP in the oxidation of HbFeZ+ and was itself oxidized to the phenoxy radical as the catalytically active molecule. However, the phenoxy radical disproportionated into 3-hydroxy-ABP and the o-quinoneimine, which was bound to the SH-groups of globin and GSH. N Hydroxy-ABP shared this metabolic fate, since nitroso-BP was also bound to the SH-groups of globin and G S H . Nevertheless, the two adducts are chemically different, with nitrosoarene sulphinamides and sulphenamides (Eyer 1985) being formed, which are readily hydrolysed by acid or base to give the arylamine (Heilmair et al. 2991, Karreth and Lenk 1991 a). With o-quinoneimines, however, thioethers are formed, which are not reduced back to the o-aminophenol on acid hydrolysis. T h e observation, that only 33% of added N-hydroxy-ABP was recovered from rat blood in nitro (Heilmair et al. 1991) as ABP and AABP, indicates that the missing 67% was bound to globin as thioethers after being oxidized to the o-quinoneimine. Acid hydrolysis would give various S-substituted o-aminophenols or odihydroxybiphenyls. Further experiments are required to detect such compounds. +
Acknowledgement The authors are grateful to Miss Renate Heilmair for competent technical assistance.
HbFe3 formation by 4-aminobiphenyl metabolites +
977
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
References EYER,P., 1985, Reactions of nitrosoarenes with sulphhydryl groups: reaction mechanism and biological significance. In Biological Oxidation of Nitrogen in Organic Molecules, edited by J . W. Gorrod and L. A. Damani (Chichester: Ellis Horwood), pp. 386-399. EYER,P., and LENGFELDER, E., 1984, Radical formation during autoxidation of 4-dimethylaminophenol and some properties of the reaction products. Biochemical Pharmacology, 33, 1005-1013. G., and WEGER,N., 1974, Reactions of 4-dimethylaminophenol EYER,P., KIESE,M., LIPOWSKY, with hemoglobin, and autoxidation of 4-dimethylaminophenol. Chemico-Biological Interactions, 8, 41-59. EVELYN, K. A., and MAI.LOY, H. T., 1938, Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. Journal of Biological Society, 128, 655-662. HEILMAIR, R., LENK,W., and STERZL, H., 1987, N-Hydroxy-N-arylacetamides. IV. Differences in the mechanism of haemoglobin oxidation in witro between N-hydroxy-N-arylacetamidesand arylhydroxylamines. Biochemical Pharmacology, 36, 2963-2972. HEILMAIR, R., KARRETH, S., and LENK,W., 1991, The metabolismof 4-minobiphenyl in rat. 11. Reaction of N-hydroxy-4-aminobiphenylwith rat blood in witro. Xenobiotica, 21, 805-81 5. HERR,F., and KIESE,M., 1959, Bestimmung von Nitrosobenzol im Blute. Naunyn-Schmiedebergs Archiv fur Experimentelle Pathologie und Pharmakologie, 235, 351-353. KARRETH, S., and LENK,W., 1991a, The metabolism of 4-minobiphenyl in rat. I. Reaction of N-hydroxy-4-aminobiphenylwith rat blood in vivo. Xenobiotica, 21, 417428. KARRETH, S., and LENK,W., 1991 b, The metabolism of 4-aminobiphenyl in rat. 111. Urinary metabolites of 4-aminobiphenyl. Xenobiotica, 21, 709-724. KIESE,M., 1974, Methemoglobinemia: A Comprehensiwe Treatise (Cleveland, Ohio: CRC Press), 260 pp. KIESE,M., REINWEIN, D., and WALLER, H. D., 1950, Kinetik der Hamiglobinbildung. IV. Mitteilung. Die Hamiglobinbildung durch Phenylhydroxylamin und Nitrosobenzol in roten Zellen in witro. Naunyn-Schmiedebergs Archiv fiir Experimentelle Pathologie und Pharmakologie, 210, 393-398. KIJNCENRERC, K., 1891, Studien iiber Oxydationen aromatischer Substanzen im thierischen Organismus. Dissertation Universitat Rostock. LENK,W., and STERZL,H., 1982, Differences in the ferrihaemoglobin-forming capabilities and carcinogenicities between monocyclic and polycyclic N-acylarylamines and their derivatives. Reviews on Drug Metabolism and Drug Interactions, IV, 171-236. LENK,W., and STERZL,H., 1987, N-Hydroxy-N-arylacetamides. 111: mechanism of haemoglobin oxidation by N-hydroxy-4-chloroactanilidein erythrocytes in witro. Xenobiotica, 17, 499-51 2. J. C., and WHITAKER, G. W., 1980, The N-hydroxylation and ringMCMAHON, R. E., TURNER, hydroxylation of 4-aminobiphenyl in witro by hepatic mono-oxygenases from rat, mouse, hamster, rabbit and guinea pig. Xenobiotica, 10. 469481. VON JACOW, R., KIESE, M., and RENNER, G., 1966, Urinary excretion of N-hydroxy derivatives of some aromatic amines by rabbits, guinea pigs and dogs. Biochemical Pharmacology, 15, 1899-1910. H., and NESTEL,K., 1967, Hydroxylamino- und Nitrosobiphenyl: Biologische OxidationsUEHLEKE, produkte von 4-Aminobiphenyl und Zwischenprodukte der Reduktion von Nitrobiphenyl. Naunym-Schmiedebergs Archiv fiir Pharmakologie und Experimenteile Pathologie, 257, 151-1 71.
1991, VOL. 21,
NO.
7, 971-977
The metabolism of 4-aminobiphenyl in rat. IV. Ferrihaemoglobin formation by 4-aminobiphenyl metabolites S. KARRETH and W. LENKT Walther Straub-Institut fur Pharmakologie und Toxikologie der LM-Universitat Miinchen, D-8000 Munchen 2, NuRbaumstr. 26, Germany
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
Received 26 March 1990; accepted 1 November 1990
1. Rats dosed with nitrosobenzene (56pmol/kg), 4-chloronitrosobenzene (53 pmol/kg), 3,4-dichloronitrosobenzene (53pmol/kg), 4-ethoxynitrosobenzene (86pmol/kg), 4nitrosobiphenyl(nitroso-BP, 55 pmollkg) or 2-nitrosofluorene (256 pmol/kg) had maximal ferrihaemoglobin (HbFe") concn of 69,68, 69,67, 55 and 42% after 15,25,48, 35, 80 and 115 min, respectively, indicating differences in solubility of the nitrosoarenes in body fluids. 2. Nitroso-BP and 3-hydroxy-4-aminobiphenyl (3-hydroxy-ABP) catalytically oxidized HbFe2' in bovine erythrocytes in vitro; nitroso-BP was three times as active as 3-hydroxy-ABP. 3',4'-Dihydroxy-4-aminobiphenyl (3',4'dihydroxy-ABP) showed only low catalytic activity, and seven other ABP metabolites exhibited only marginal activity. 3. Nitroso-BP was inactive in solutions of purified human Hb, but 3-hydroxy-ABP catalytically oxidized HbFe", indicating that nitrosoarenes oxidize HbFe'' in erythrocytes in vitro and in oivo by a mechanism different from that of o-aminophenols. T h e second-order rate constant for HbFe*+ oxidation by 3-hydroxy-ABP at 37°C was k2=19.1f1~31/molper s.
Introduction If ferrihaemoglobin (HbFe3+) is formed in vivo after administration of an arylamine which does not form HbFe3+ in vitro, HbFe3+ formation is due to active metabolites produced by N- and/or ring-hydroxylation in the liver. T h e role which arylhydroxylamines and aminophenols play during HbFe3+ formation in vivo merits investigation because HbFe3'-forming activity is a further aspect of drug metabolism which has consequences for the acute or chronic toxicity of the arylamine. 4-Aminobiphenyl (ABP), for example, the strong HbFe3+-forming activity of which was first observed by Klingenberg (1891) in dog, is metabolized by rat liver enzymes in vitro to N-hydroxy-4-aminobiphenyl(N-hydroxy-ABP), 3-hydroxy-4aminobiphenyl(3-hydroxy-ABP),2'-hydroxy-4-aminobiphenyl(2'-hydroxy-ABP) and 4'-hydroxy-4-aminobiphenyl(4-hydroxy-ABP) (McMahon et al. 1980). After injection of ABP into dogs, rabbits and cats, N-hydroxy-ABP was found in the blood (von Jagow et al. 1966, Uehleke and Nestel 1967), as a sensitive assay (Herr and Kiese 1959) was available. Because no such method was available for the phenolic metabolites, these were not determined, and therefore it remained unknown whether or not they play any role during ABP-induced HbFe3+ formation in vivo. T h e HbFe3+-forming activity of N-hydroxy-ABP, in comparison with other arylhydroxylamines, was studied in rat (Lenk and Sterzll982) and in vitro (Heilmair et al. 1987, Lenk and Sterzl 1987); we now report studies of the HbFe3+-forming activity of 4-nitrosobiphenyl (nitroso-BP) in comparison with other relevant nitrosoarenes in vivo and of 3-hydroxy-ABP and other metabolites of ABP in comparison with N-hydroxy-ABP in vitro.
7 Author to whom correspondence should be addressed. 0049-8254/91 $3.00 0 1991 Taylor & Francis Ltd
972
S. Karreth and W. Lenk
Materials and methods
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
Chemicals Nitrosoarenes. Commercial nitrosobenzene was recryst. from ethanol prior to use. 4-Nitrosobiphenyl was obtained by oxidation of N-hydroxy-4-aminobiphenylwith periodate as described (Karreth and Lenk 1991 a); U.V.absorption in methanol showed A,,, = 341 and 231 nm. 2-Nitrosofluorene was prepared from 2-nitrofluorene according to von Jagow et al. (1966); the crude product was purified by column chromatography and crystn from n-hexane and showed A,, = 357, 260 and 240 nm in methanol. Other nitrosoarenes were obtained from the corresponding arylhydroxylamines by oxidation with FeCI,, steam distillation of the precipitate and recrystn from small volumes of n-heptane or ethanol at -27°C; U . V . absorption in methanol, 4-chloronitrosobenzene, A,,= 313, 288 and 223 nm; 3,4-dichloronitrosobenzene, A,, = 31 1, 289 and 231 nm; 4-ethoxynitrosobenzene, A,, = 341 and 239 nm. Derivatives of 4-aminobiphenyl. Preparation and properties of 3-hydroxy-, 2'-hydroxy-, 4'-hydroxy- and 3',4-dihydroxy-4-aminobiphenyl,3-hydroxy-, 2'-hydroxy-, 4-hydroxy- and 3',4-dihydroxy-4acetylaminobiphenyl, 3',4'-dihydroxy-4-nitrobiphenyl and 4-(4-aminophenyl)-l,2-henzoquinoneare described elsewhere (Karreth and Lenk 1991b). Biological material Bovine blood was obtained from the slaughter-house and mixed with heparin to prevent clotting. Purified human haemoglobin A was prepared from outdated human blood concentrate according to Heilmair et al. (1987). Animals Female Sprague-Dawley rats of 20&300g body wt were housed in Acme stainless steel metabolism cages, and fed Alma Standard diet for mice and rats (0801-H 10031, D-8960 (Kempten), with water ad libitum. Methods Dosing rats with nitrosoarenes. Nitrosoarenes were finely ground, suspended in 0.25% agar soln (prepared from 0.9% saline) and injected i.p. in rats. At the times indicated, 3 drops of blood were withdrawn from the tail vein for the determination of HhFe3' and total IIbFe''. Bovine blood was separated into plasma and packed erythrocytes by centrifugation at +4"C. T h e packed erythrocytes were washed three times with 0.9% saline and resuspended in Krebs-Ringer phosphate buffer pH 7.4 without additives. Addition ojsubstrotes. After shaking suspensions of red cells or solns of purified haemoglobin at 37°C in a water-bath for 5 min, methanolic solns of the substances were added and aliquots of 0.1 ml removed at times indicated. HbFe3' concentration and total HbFe2' in rat blood, suspensions of bovine erythrocytes or s o h of purified haemoglobin were determined according to Evelyn and Malloy (1938) and modified by Kiese (1974). Aliquots of 0.1 ml blood (in duplicate) or red cell suspension were haemolysed with lOml ice-cold water, 1 ml 0.2 M Sorensen buffer pH 6.6 added, centrifuged and the light absorption of the clear supernatant was measured at 550nm.
Results Ferrihaemoglobin formation in rats by six nitrosoarenes Following i.p. injection of nitrosobenzene (NOB), 4-chloronitrosobenzene (4-CINOB), 3,4-dichloronitrosobenzene (3,4-CI2NOB), 4-ethoxynitrosobenzene (4-ENOB), 4-nitrosobiphenyl (4-NOBP) and 2-nitrosofluorene (2-NOF), HbFe3 concns in rat blood were monitored for 5 h (figure 1). In the first phase of the reaction with HbFe", a time-dependent increase in HbFe3+ concn occurred which lasted as long as HbFe3+ formation exceeded enzymic HbFe3+ reduction. However, when the reactive nitroso compounds were inactivated by metabolism, HbFe3+ formation ceased and HbFe3+ concn passed its maximum. Thereafter HbFe3+ reduction decreased the concn of HbFe3+ in the second phase of the reaction. As can be seen from figure 1, the nitrosoarenes differed in the initial velocity of HbFe3+ formation, both in the time and magnitude of the maximal HbFe3+ concn, and in the subsequent decline of the HbFe3+ concn. +
HbFe3+ formation by 4-aminobiphenyl metabolites
973
90 -
0
z
70
50
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
30
10 1
2
3
4
5
Time a f t e r d o s i n g ( h ) Figure 1.
Kinetics of ferrihaemoglobinformation in oiwo after i.p. injection of various nitrosoarenes to female rats.
Rats were dosed with nitrosobenzene (56pmol/kg, -A-),
4-chloronitrosobenzene (53 pmol/kg,
-0-), 3,4-dichloronitrosobenzene(53 pmollkg, -0-),4-ethoxynitrosobenzene (86 pmol/kg,
-Ap), 4-nitrosobiphenyl (54.6pmol/kg, -0-) and 2-nitrosofluorene (256pmol/kg, -B-). For i.p. injection, the compounds were finely ground, suspended in 0.25%agar s o h , and injected.
Symbols indicate mean values from three animals each.
Due to differences in solubility in body fluids, such as lymph and blood, the four monocyclic nitroso compounds were active from the first minute, whereas nitrosoBP and 2-NOF became active after a lag phase of 5 min. Differences in the initial velocity of HbFe3+ formation, of 6.7%/min for NOB, 55%/min for 4-ClNOB, 3.3%/min for 3,4-C12NOB, 3.0%/min for 4-ENOB, 1.4%/min for nitroso-BP and 0.6%/min for 2-NOF showed, however, that differences in solubility exist also among the monocyclic nitroso compounds, although these were not as marked as between the monocyclic and polycyclic analogues. Maximal HbFe3 concns of 69% produced by 56pmol/kg NOB were determined after 15 min, 67% HbFe3+ by 53 pmol/kg 4-ClNOB after 25 min, 69% HbFe3+ by 53 pmol/kg, 3,4-C12NOB after 45min, 67% HbFe3+ by 86pmollkg 4-ENOB after 35min, 55% HbFe3+ by 546 pmol/kg nitroso-BP after 75 min, and 42% HbFe3+ only by 256pmol/kg 2NOF after 110 min. Thereafter HbFe3+ concn declined most rapidly at a rate of 24%/h after dosing rats with NOB, followed by 4-ENOB at a rate of 18%/h,while the others produced a decline at a rate of 14%/h. This is an indication that metabolism and HbFe3+ reduction are related to each other, since both NOB and 4-ENOB are rapidly metabolized in rats. HbFe3' formation by 2-NOF was sluggish and inefficient, because a five-fold higher dose produced a maximum of 42% HbFe3+ only. T h e reason for the ineffective and protracted HbFe3+ formation may be found in the low solubility of 2-NOF in lymph and blood and in its incomplete mobilization from the peritoneal cavity, as residual 2-NOF was found remaining in the peritoneal cavity some days later. +
S . Karreth and W . Lenk
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
974
Ferrihaemoglobin formation in suspensions of bovine erythrocytes Suspensions of washed bovine erythrocytes (HbFe2+concn: 5 mM) incubated at 37°C with 110, 50 or 1 O p nitroso-BP, ~ oxidized 26, 63 and 150equiv. of H b F e 2 + , respectively, in 1 h. After 2 h, 33, 71 and 215 equiv. of H b F e 2 + , respectively, were oxidized (figure 2). This is an indication that nitroso-BP oxidized H b F e 2 + catalytically and that the catalytic activity increased with an increasing ratio of nitrosoarene : HbFe2 +. This finding is in agreement with the mechanism of haemoglobin oxidation in erythrocytes in vivo and in vitro by arylhydroxylamines (Kiese et al. 1950, Heilmair et al. 1991). In suspensions of washed bovine erythrocytes (HbFe2+ concn: 5 mM) incubated at 37°C with 1OmM 3-hydroxy-ABP, H b F e 3 + was rapidly formed, and within 1 h H b F e 2 +was completely oxidized (figure 3). With 1-23mM 3-hydroxy-ABP the rate of HbFe3+ formation was the same, but the reaction was finished after 45 min, when 91% HbFe3+ was formed. Similarly 93 p M 3-hydroxy-ABP oxidized 8.6 equiv. of HbFe’ in 15 min, 28 equiv. after 1 h and 39 equiv. after 2 h; 50 p~ 3-hydroxy-ABP oxidized 4.7equiv. of HbFe” after 15 min, 21 equiv. after 1 h, and 49 equiv. after 2 h, indicating that the catalytic activity of 50 PM 3-hydroxy-ABP was only one-third of the catalytic activity of nitroso-BP. On incubation at 37°C of suspensions of washed bovine erythrocytes (HbFe” concn: 5 mM) with 10mM 3‘,4’-dihydroxy-ABP, 62% HbFe3+ was formed after 1 h, 85% after 2 h and 100%after 3 h, i.e. each 3’,4’-dihydroxy-ABP had oxidized 0.3,0.4 and 0.5 equiv. of H b F e 2 + ,respectively; with 5 mM 3’,4’-dihydroxy-ABP, each mmol oxidized 0.6, 0 8 and 0.95 equiv. of H b F e 2 + , and with 1 m M 3’,4’-dihydroxy-ABP, each mmol oxidized 1.3 equiv. of HbFe2+ in 1 h, 2.0 equiv. in 2 h and 2.5 equiv. in 3 h, indicating low catalytic activity of 3’,4’-dihydroxy-ABP at a molar ratio of 1 : 5 . 3-Hydroxy-AABP, 2’-hydroxy-ABP, 2’-hydroxy-AABP, 4’-hydroxy-ABP, 4’hydroxy-AABP, 3’,4‘-dihydroxy-AABP and 4-(4-aminophenyl)-l,2-benzoquinone, which were also examined for their HbFe3+-forming activity in suspensions of bovine erythrocytes, exhibited only marginal activity and lacked catalytic activity. +
Ferrihaemoglobin formation in solutions of purified haemoglobin On incubation of purified haemoglobin (HbFe2+ concn: 5.1 mM) at 37°C with 6 7 p nitroso-BP ~ for 3.5 h, 8.9% HbFe3+ was formed, indicating that not more HbFe3+ was formed than was produced by autoxidation of HbO,. This proves the inability of the nitrosoarene to oxidize H b F e 2 + , unless it is activated by enzymic reduction to the arylhydroxylamine. On incubation of purified haemoglobin (HbFe” concn: 5.1 mM) at 37°C with 6 7 p 3-hydroxy-ABP, ~ 41% HbFe3+ was formed in 1.5 h, i.e. each 3-hydroxy-ABP has oxidized 24 equiv. of HbFe*+. In contrast to nitroso-BP, the o-aminophenol displayed catalytic activity, which shows that the mechanism of HbFe” oxidation by o-aminophenols differs from that of nitrosoarenes. T h e order of the reaction of 3-hydroxy-ABP with H b F e 2 + was determined by measuring initial velocities of HbFe3+ formation at a constant concn of HbFe” (155 p ~ with ) varying concns of 3-hydroxy-ABP (5-100 PM), and constant concn of 3-hydroxy-ABP ( 1 0 0 ~ with ~ ) varying concns of H b F e 2 + ( 1 3 0 p ~ - lmM). O n plotting initial velocities of HbFe3+ formation versus concn of either H b F e 2 + or 3hydroxy-ABP in a double-logarithmic scale, two parallel straight lines with a slope of 1 were obtained, indicating that haemoglobin oxidation by 3-hydroxy-ABP was first order with respect to either reactant, and second order with respect to HbFe3+
HbFe3 formation by 4-aminobiphenyl metabolites
975
+
0
5!
roo
X L
\
50
0
10pM
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
m Time of incubation (h) Figure 2.
Kinetics of ferrihaemoglobin formation in erythrocytes in witro by 4-nitrosobiphenyl.
Suspensions of washed bovine erythrocytes (HbFe” concn: 5 mM) in Krebs-Ringer phosphate buffer, pH 7.4, were incubated at 37°C with various concns of nitroso-BP and HbFe3+ determined at times indicated. Symbols are from single experiments.
0
z
100
X
60
20 2
3
5
L
Time of incubation(h) Figure 3.
Kinetics of ferrihaemoglobin formation in erythrocytes in witro by 3-hydroxy-4aminobiphenyl.
Suspensions of washed bovine erythrocytes (HbFe” concn: 5 mM) in Krebs-Ringer phosphate buffer, pH7.4, were incubated at 37°C with various concns of 3-hydroxy-ABP and HbFe” determined at times indicated. Symbols are from single experiments.
formation. Based on these results from table 1, the second-order rate constant was calculated to be k2 = 19.1& 1.3 I/mol per s.
Discussion The results presented here show that nitroso-BP catalytically oxidized HbFe2+ in erythrocytes, but was inactive in solutions of purified haemoglobin. In contrast, 3-hydroxy-ABP catalytically oxidized HbFe2+both in erythrocytes and in solutions of haemoglobin, indicating differences in the mechanism of HbFe’ oxidation between these two classes of compounds. Kiese et al. (1950) recognized that nitrosoarenes require enzymic reduction to the active arylhydroxylamine for the oxidation of many equiv. of HbFe” in erythrocytes in vivo and in vitro. Therefore in solutions of purified haemoglobin nitrosoBP cannot be reduced to the active arylhydroxylamine. +
S. Karreth and W . Lenk
976
Table 1. The rate coefficient of haemoglobin oxidation by 3-hydroxy-4-aminobiphenyldetermined from initial velocities. 3-Hydroxy-4-aminobiphenyl
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
(mM)
Initial HhFe” (mM)
Formation of HhFe3’ (mM/s)
k2
(limo1 per
s)
0.1 0.05 0.0 1 0.005
0.155 0.155 0155 0.1 55
0.24 x 10-37 0 1 4 10-3 ~ 0.03 x 10-3 002 x 10-3
15.5 18.1 25.8 25.8
0.1 0.1 0.1 0.1 0.1
1.0 0.45 0.291 0.223
1.87 x 10-31 077 x 0.51 x 10-3 0.40 x 10-3 0 . 2 0 ~10-3
18.7 17.1 17.5 17.9 15.4 19.1 5 1.3 (SEM)
0.13
t Autoxidation of Hb: 0,155mM HhFe”: 0 0 8 6 p ~ / m i n . $Autoxidation of Hb: 1 mM HbFe”: @ 8 6 p ~ / m i n . 0 1 3 0 m ~HbFeZt: 0.152p~lrnin. It was demonstrated that aminophenols catalytically oxidize HbFe’ in red cells in nitro by Eyer et al. (1974) with 4-dimethylaminophenol (DMAP). At a molar ratio of DMAP : HbFe2+of 1 :42, each mol of DMAP oxidized an average of 60 equiv. of HbFe”, and its catalytic activity depended on the molar ratio of DMAP : HbFe”. Experiments with l4C-labe1led DMAP have shown that all activity was bound to the globin moieties. T h e mechanism of catalytic oxidation of HbFeZ+by DMAP was elucidated by Eyer and Lengfelder (1984). DMAP was oxidized by the activated oxygen of HbO, to the phenoxy radical via one-electron transfer, and the phenoxy radical in turn oxidizes HbFe2+ and is reduced back to DMAP. But the phenoxy radical also disproportionates into the quinoneimine and DMAP, and the quinoneimine is bound to the SH-groups of globin (and G S H in erythrocytes) to give thioethers. Any 3-hydroxy-ABP, which was produced in the liver of rats after dosing with ABP, therefore, participated with N-hydroxy-ABP in the oxidation of HbFeZ+ and was itself oxidized to the phenoxy radical as the catalytically active molecule. However, the phenoxy radical disproportionated into 3-hydroxy-ABP and the o-quinoneimine, which was bound to the SH-groups of globin and GSH. N Hydroxy-ABP shared this metabolic fate, since nitroso-BP was also bound to the SH-groups of globin and G S H . Nevertheless, the two adducts are chemically different, with nitrosoarene sulphinamides and sulphenamides (Eyer 1985) being formed, which are readily hydrolysed by acid or base to give the arylamine (Heilmair et al. 2991, Karreth and Lenk 1991 a). With o-quinoneimines, however, thioethers are formed, which are not reduced back to the o-aminophenol on acid hydrolysis. T h e observation, that only 33% of added N-hydroxy-ABP was recovered from rat blood in nitro (Heilmair et al. 1991) as ABP and AABP, indicates that the missing 67% was bound to globin as thioethers after being oxidized to the o-quinoneimine. Acid hydrolysis would give various S-substituted o-aminophenols or odihydroxybiphenyls. Further experiments are required to detect such compounds. +
Acknowledgement The authors are grateful to Miss Renate Heilmair for competent technical assistance.
HbFe3 formation by 4-aminobiphenyl metabolites +
977
Xenobiotica Downloaded from informahealthcare.com by University of Auckland on 10/28/14 For personal use only.
References EYER,P., 1985, Reactions of nitrosoarenes with sulphhydryl groups: reaction mechanism and biological significance. In Biological Oxidation of Nitrogen in Organic Molecules, edited by J . W. Gorrod and L. A. Damani (Chichester: Ellis Horwood), pp. 386-399. EYER,P., and LENGFELDER, E., 1984, Radical formation during autoxidation of 4-dimethylaminophenol and some properties of the reaction products. Biochemical Pharmacology, 33, 1005-1013. G., and WEGER,N., 1974, Reactions of 4-dimethylaminophenol EYER,P., KIESE,M., LIPOWSKY, with hemoglobin, and autoxidation of 4-dimethylaminophenol. Chemico-Biological Interactions, 8, 41-59. EVELYN, K. A., and MAI.LOY, H. T., 1938, Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. Journal of Biological Society, 128, 655-662. HEILMAIR, R., LENK,W., and STERZL, H., 1987, N-Hydroxy-N-arylacetamides. IV. Differences in the mechanism of haemoglobin oxidation in witro between N-hydroxy-N-arylacetamidesand arylhydroxylamines. Biochemical Pharmacology, 36, 2963-2972. HEILMAIR, R., KARRETH, S., and LENK,W., 1991, The metabolismof 4-minobiphenyl in rat. 11. Reaction of N-hydroxy-4-aminobiphenylwith rat blood in witro. Xenobiotica, 21, 805-81 5. HERR,F., and KIESE,M., 1959, Bestimmung von Nitrosobenzol im Blute. Naunyn-Schmiedebergs Archiv fur Experimentelle Pathologie und Pharmakologie, 235, 351-353. KARRETH, S., and LENK,W., 1991a, The metabolism of 4-minobiphenyl in rat. I. Reaction of N-hydroxy-4-aminobiphenylwith rat blood in vivo. Xenobiotica, 21, 417428. KARRETH, S., and LENK,W., 1991 b, The metabolism of 4-aminobiphenyl in rat. 111. Urinary metabolites of 4-aminobiphenyl. Xenobiotica, 21, 709-724. KIESE,M., 1974, Methemoglobinemia: A Comprehensiwe Treatise (Cleveland, Ohio: CRC Press), 260 pp. KIESE,M., REINWEIN, D., and WALLER, H. D., 1950, Kinetik der Hamiglobinbildung. IV. Mitteilung. Die Hamiglobinbildung durch Phenylhydroxylamin und Nitrosobenzol in roten Zellen in witro. Naunyn-Schmiedebergs Archiv fiir Experimentelle Pathologie und Pharmakologie, 210, 393-398. KIJNCENRERC, K., 1891, Studien iiber Oxydationen aromatischer Substanzen im thierischen Organismus. Dissertation Universitat Rostock. LENK,W., and STERZL,H., 1982, Differences in the ferrihaemoglobin-forming capabilities and carcinogenicities between monocyclic and polycyclic N-acylarylamines and their derivatives. Reviews on Drug Metabolism and Drug Interactions, IV, 171-236. LENK,W., and STERZL,H., 1987, N-Hydroxy-N-arylacetamides. 111: mechanism of haemoglobin oxidation by N-hydroxy-4-chloroactanilidein erythrocytes in witro. Xenobiotica, 17, 499-51 2. J. C., and WHITAKER, G. W., 1980, The N-hydroxylation and ringMCMAHON, R. E., TURNER, hydroxylation of 4-aminobiphenyl in witro by hepatic mono-oxygenases from rat, mouse, hamster, rabbit and guinea pig. Xenobiotica, 10. 469481. VON JACOW, R., KIESE, M., and RENNER, G., 1966, Urinary excretion of N-hydroxy derivatives of some aromatic amines by rabbits, guinea pigs and dogs. Biochemical Pharmacology, 15, 1899-1910. H., and NESTEL,K., 1967, Hydroxylamino- und Nitrosobiphenyl: Biologische OxidationsUEHLEKE, produkte von 4-Aminobiphenyl und Zwischenprodukte der Reduktion von Nitrobiphenyl. Naunym-Schmiedebergs Archiv fiir Pharmakologie und Experimenteile Pathologie, 257, 151-1 71.
