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Mutagenicity of diphenylhydantoin and some of its metabolites towards Salmonella typhimurium strains
103 (1982)219-228 Elsevier BiomedicalPress
Mutation Research,
219
Mutagenicity of diphenylhydantoin and some of its metabolites towards Salmonella typhimurium strains P. Sezzano b, A. Raimondi b, M. Arboix c and C. P a n t a r o t t o a,* a istituto di Ricerche Farmacologiche "Mario Negri" Via Eritrea 62, 20157 Milan (Italy), a lstituto di Microbiologia, Universit~ di Milano, Via Mangiagalli 31, 20133-Milan (Italy) and c Departamento de Farmacologia, Universidad Autonoma de Barcelona, Bellaterra, Barcelona (Spain)
(Accepted 3 August 1981)
Summary The mutagenicities of 5,5-diphenylhydantoin (DPH) and its major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin(HPPH) were tested in vitro using different Salmonella strains (TA1535, TA100, TA1537, TA1538, TA98). Experiments were carried out at various concentrations in the absence and in the presence of an activating system consisting of hepatic $9 fraction from control rats and from rats pretreated with phenobarbital (PB), /3-naphthoflavone (BNF), 3-methylcholanthrene (3-MC) and Aroclor 1254 (PCB). DPH slightly increased the number of revertants per plate only after incubations with TA1538 in the presence of the $9 fraction from the liver of 3-MC- and PCBpretreated animals. A similar but more significant frameshift mutation was observed for HPPH on both TA98 and TA1538 strains and in conditions of metabolic activation by the liver microsomal fractions of rats after pretreatment with BNF, 3-MC and especially PCB. Parallel experiments on the metabolism of DPH to HPPH and of HPPH to the catechol derivative in vitro support the hypothesis of an involvement of epoxide intermediates in the mutagenic activity of DPH.
5,5-Diphenylhydantoin (DPH) is widely used in clinical practice for the treatment of epilepsy (Laidlaw and Richens, 1976; Johannessen et al., 1980). Neurological, hematological, immunological and hepatic toxicity in subjects under therapy with DPH has been reported by several authors (Glaser, 1972; Dam, 1972; Klein et al., 1976; Dukes, 1980), and the possibility of this drug having teratogenic and carci*To whomcorrespondenceshould be addressed. 0165-7992/82/0000-0000/$02.75 © ElsevierBiomedicalPress
220 nogenic effects has also been discussed (Loughman et al., 1973; De Vore and Woodbury, 1977; Editorial, 1971; Kruger and Harris, 1972). Some of these toxicities might be related to DPH metabolism through the formation of chemically reactive intermediates. Reports in the literature show that DPH is metabolized to phenolic, dihydrodiol and catechol derivatives and discuss the possible intermediacy of epoxides (Glazko, 1973). This hypothesis has been supported by recent results from our group on irreversible DPH binding to rat-liver microsomal proteins catalyzed by a mono-oxygenase dependent on cytochrome P-448 (Pantarotto et al., 1981). It is known that epoxide intermediates produced during the biotransformation of exogenous compounds are responsible for their mutagenic effects. Therefore it seemed interesting to us to study the mutagenicity of DPH and HPPH in vitro and in conditions of controlled metabolism.
Materials and methods
Chemicals. DPH, HPPH and 5-(4-methylphenyl)-5-phenylhydantoin (MPPH) were obtained from Aldrich, Beerse (Belgium). 5-(3,4-Dihydroxyphenyl)-5-phenylhydantoin (DHP), as reference material, was extracted with chloroform at pH 4.5 from the urine of DPH-treated rats and first purified by preparative thin-layer chromatography on silica-gel glass plates developed in a solvent system consisting of chloroform : methanol : acetic acid, 90:10 : 1 (v/v). The Rf value of DHP was 0.37. Further purification was achieved by high-performance liquid chromatography on a reversed-phase column (0.25 m long × 2.6 mm i.d.) packed with 10 #m (average particle diameter) LiChrosorb RP-18 (Merck, Darmstadt, West-Germany). The column was eluted with a mixture of acetonitrile and acetate buffer, 0.02 M, pH 4.5 (40: 60, v/v). PB was obtained from Merck, Darmstadt (West-Germany); 3-MC from Sigma, St. Louis, MO (U.S.A.); BNF from Aldrich, Beerse (Belgium); and PCB from Analabs, North Haven, CN (U.S.A.). All reagents were analytical grade. Animals. Male CD-COBS rats (200-220 g, b.w.) from Charles River Italy (Calco, Como, Italy) were used. Hepatic microsomal cytochrome P-450/448 was induced with PB (40 mg/kg i.p., in 0.9% saline, twice daily for 3 days), BNF (60 mg/kg i.p., in corn oil, once daily for 2 days), 3-MC (40 mg/kg i.p., in corn oil, once daily for 3 days) or PCB (500 mg/kg i.p., in corn oil, once). All rats were fasted for 24 h before they were killed. Preparation o f liver 9000 × g supernatant fractions. Groups of 5 pretreated rats were used for the preparation of each batch of liver 9000 × g supernatant fractions ($9). Control rats and rats pretreated with PB, BNF and 3-MC were killed by decapitation 24 h after the last treatment, and PCB-pretreated animals were killed after 5 days. The livers were excised and pooled, and $9 fractions were obtained
221
according to the procedure of Ames et al. (1975). Proteins were determined by the method of Lowry et al. (1951) with albumin fraction V as the standard. Mutagenicity assay. Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 were used. The mutagenicity test was carried out in airtight petri dishes according to the method of Ames et al. (1973). DPH and H P P H were dissolved in dimethyl sulfoxide (DMSO) and added at 45 °C to 2 ml of molten top agar with added L-histidine. HC1 (0.5 mM) and biotin (0.5 mM) together with 0.1 ml of an overnight nutrient broth culture of the bacterial tester strains and, if required, 0.5 ml of $9 fraction fortified with an NADPH-regenerating system ($9 mix). Samples were plated on Vogel-Bonner minimal medium dishes and incubated at 37 °C for 48 h. After incubation, the number of histidine-revertant colonies was determined.
Gas chromatographic-mass fragmentographic determination of HPPH and DHP H P P H and DHP were determined according to Arboix and Pantarotto (1981). The incubation medium was resuspended in 6 ml of a 0.3 M NaH2PO4.H20 aqueous solution containing 500 ng of MPPH as internal standard. Hermetically sealable glass tubes bearing a Teflon disk in the inner part of the cap were used, and the samples were extracted with 5 ml of 0.5 M trideuteromethyl iodide methylene chloride solution after addition of 0.5 ml of 10 N NaOH and 100 /~1 of 0.1 M tetrahexylammonium hydrogen sulfate solution in 0.1 N NaOH. Extractions were carried out for 2 h by shaking at 60 °C in subdued light. The organic phase (4.5 ml) was transferred to conical glass tubes and evaporated to dryness under a gentle stream of nitrogen in a water-bath at 35 °C. The residue was resuspended in 50/~1 of methylene chloride, and the tetrahexylammonium salt was precipitated by addition of 2 ml of n-hexane. The tubes were capped and centrifuged for 5 min at 4000 x g. The supernatant fraction (1.8 ml) was transferred to conical glass tubes and evaporated to dryness under a nitrogen stream at 35 °C. The residue was dissolved in 100 #1 of Tri-Deuter-8® solution (Pierce, Rockford, IL, U.S.A.), and a 1 - 5 / d portion of this solution was injected for mass-fragmentographic analysis. Tri-Deuter-8® was used to achieve complete derivatization of DHP. For mass fragmentography the instrument (LKB 2091 mass spectrometer equipped with a computer system model 2130 for data acquisition and calculation) was focused on the ions at m/e 300 (molecular ion in the spectrum of MPPH ditrideuteromethyl derivative), 319 (molecular ion in the spectrum of H P P H tritrideuteromethyl derivative) and 352 (molecular ion in the spectrum of DHP tetratrideuteromethyl derivative).
Results
DPH was tested for mutagenicity in various Salmonella typhimurium strains
222 TABLE 1 MUTAGENICITY OF DPH FOR 5 SALMONELLA STRAINS DHP concentration ~g/plate) 0 1 10 l~ 1000
Number of His÷ revertant colonies TA100
TA1535
TA98
TA1537
TA1538
136± 8 133± 6 124±10 130± 4 136± 9
16.0±1.9 10.6±1.4 16.2±1.8 17.0±0.5 14.5±1.8
~.2±2.2 28.0±2.3 21.2±2.8 23.3±1.7 20.5±3.7
6.7±1.2 5.6±0.6 8.0±1.0 7.0±2.0 8.3±2.0
13.5±1.6 17.0±2.3 14.7±1.7 11.9±2.5 13.5±1.0
Each value is the mean ± S,E. of 5 determinations.
(TA100, T A 1 5 3 5 , T A 9 8 , T A 1 5 3 7 , TA1538), a n d the results o f these e x p e r i m e n t s are r e p o r t e d in T a b l e 1. T h e d r u g was n o t m u t a g e n i c at a n y o f the c o n c e n t r a t i o n s tested, r a n g i n g f r o m 1 to 1000/~g/plate. S i m i l a r results were o b t a i n e d with H P P H at the s a m e c o n c e n t r a t i o n s ( T a b l e 2). I n b o t h e x p e r i m e n t s , cell survival was 100°70 indic a t i n g t h a t the d r u g a n d its h y d r o x y l a t e d m e t a b o l i t e were n o t c y t o t o x i c even a t doses n e a r the limit o f their solubility. T a b l e s 3 a n d 4 s h o w the m u t a g e n i c i t y o f D P H a n d H P P H w h e n i n c u b a t i o n s were c a r r i e d o u t in the presence o f $9 mix f r o m the livers o f c o n t r o l rats a n d rats pret r e a t e d with d i f f e r e n t c y t o c h r o m e P - 4 5 0 / 4 4 8 i n d u c e r s . T h e c a p a c i t y o f $9 mix p r e p a r a t i o n s to a c t i v a t e a p r o m u t a g e n was tested with b e n z o [ a ] p y r e n e at a dose o f 5 /zg/plate. W i t h $9 mix f r o m P C B - p r e t r e a t e d rats, a 7-fold i n c r e a s e in the n u m b e r o f s p o n t a n e o u s r e v e r t a n t colonies was o b s e r v e d . A f t e r m e t a b o l i c a c t i v a t i o n by the $9 mix f r o m the livers o f 3 - M C - a n d P C B - p r e t r e a t e d rats, D P H at c o n c e n t r a t i o n s , respectively, o f 25 a n d 250 # g / p l a t e , h a d limited m u t a g e n i c activity o n l y t o w a r d s T A 1 5 3 8 . M o r e significant results were o b s e r v e d for H P P H w h i c h a l s o p r o v e d to be a slight f r a m e s h i f t m u t a g e n . Statistically significnt d a t a (p < 0.01) were o b t a i n e d w h e n H P P H was tested on strains T A 9 8 a n d T A 1 5 3 8 at the r e p o r t e d c o n c e n t r a t i o n s a n d in the presence o f $9 mix f r o m rats p r e t r e a t e d with B N F , 3 - M C a n d especially
TABLE 2 MUTAGENICITY OF HPPH FOR 5 SALMONELLA STRAINS HPPH concentration ~g/plate)
Number of His + revertant colonies TA100
TA1535
TA98
TA 1537
TA1538
0 1 10 1~ 1000
140±12 128± 2 139±10 132±11 122±14
11.2±0.5 16.0±2.2 13.3±1.7 12.0±1.8 14.0±2.3
20.0±2.0 21.0±2.1 21.6±1.6 23.0±4.0 27.0±4.5
7.0±1.1 8.1±2.0 10.0±1.4 7.6±1.6 8.3±1.8
11.0±2.1 15.3±0.8 11.3±2.9 12.8±1.1 13.0±1.0
Each value is the mean ± S.E. of 5 determinations.
223
TABLE 3 MUTAGENICITY OF DPH FOR 5 SALMONELLA STRAINS IN THE PRESENCE OF $9 MIX OBTAINED FROM THE LIVERS OF CONTROL RATS AND RATS PRETREATED WITH DIFFERENT CYTOCHROME P-450/448 INDUCERS DPH concentration (~g/plate)
Number of His + revertant colonies TA100
TA1535
TA98
TA1537
TA1538
Saline
0 25 250
129± 1 134± 6 130± 2
14.3±0.6 15.0±1.4 11.0±2.1
26.1±1.8 22.3±1.3 20.0±2.4
9.0±1.1 7.2+0.8 11.0±1.3
11.0±2.1 12.3±1.6 13.6±2.3
Corn oil
0 25 250
140± 8 137±12 129± 7
13.7±0.2 9.9±2.3 15.3±1.6
26.6±0.5 21.0±1.2 27.3±2.7
10.3±1.2 8.7±0.6 7.4±1.3
13.7±0.2 17.1±0.9 15.0±1.4
PB
0 25 250
130± 5 139± 2 126± 3
8.9±2.3 13.5±0.2 11.3±1.2
19.7±3.0 25.4±0.5 21.9±1.7
8.6±1.7 6.5±0.5 7.3+2.8
12.5±2.0 11.2±1.1 14.6+2.0
BNF
0 25 250
127± 5 135± 2 140± 6
14.2±2.0 12.6±0.3 15.3±2.1
25.2±2.4 22.3±2.6 23.0±0.8
8.9±0.4 13.0±0.5 14.3±2.7
14.7+1.7 20.0±2.3 23.0+2.6
3-MC
0 25 250
120± 6 135± 1 138± 4
10.8±1.2 12.6±0.3 9.6±0.3
20.4±2.3 33.0±3.5 36.5±2.0
7.8±1.6 9.0±0.5 11.5±1.4
13.4±0.8 31.6±0.3 a 16.5±1.5
PCB
0 25 250
141± 4 141±12 137± 4
14.6±2.0 13.3±0.8 11.6±1.4
20.4±3.2 37.3±2.1 26.1±3.2
9.6±1.3 11.6±2.0 11.0±1.6
13.7±1.2 27.0+1.1 ~.8±2.6 a
$9 mix
Each value is the mean +_S.E. of 5 determinations. a Only values significantly different (p < 0.01) and at least double the number of spontaneous revertants are considered significant for mutagenicity.
P C B . A t the dose o f 1000 # g / p l a t e , D P H and H P P H m u t a g e n i c i t y was no l o n g e r detectable, p r o b a b l y because o f a substrate i n h i b i t o r y e f f e c t o n d r u g - m e t a b o l i z i n g enzymes. This h y p o t h e s i s is s u p p o r t e d by q u a n t i t a t i v e e v a l u a t i o n o f D P H an d H P P H m e t a b o l i s m , s h o w n in T a b l e s 5 a n d 6. D e t e r m i n a t i o n s w e r e car r i ed o u t in the s a m e i n c u b a t i o n m e d i a as f o r the m u t a g e n i c i t y test. Th e d a t a indicate t h a t H P P H an d D H P f o r m a t i o n f r o m D P H a n d H P P H , respectively, is c a t a l y z e d by a c y t o c h r o m e P - 4 4 8 - d e p e n d e n t m o n o - o x y g e n a s e , these h y d r o x y l a t i o n s being i n d u ced by B N F , 3M C a n d P C B b u t n o t by P B . T h e f a il u r e to d e t e r m i n e D H P af t er i n c u b a t i o n s with D P H m i g h t be related to the limit o f sensitivity o f o u r m a s s - f r a g m e n t o g r a p h i c proc e d u r e w h i c h in these c o n d i t i o n s was 50 n g / s a m p l e .
224 TABLE 4 M U T A G E N I C I T Y OF H P P H FOR 5 SALMONELLA STRAINS IN T H E PRESENCE OF $9 MIX O B T A I N E D FROM T H E LIVERS OF CONTROL RATS AND RATS PRETREATED W I T H DIFFERENT C Y T O C H R O M E P-450/448 INDUCERS $9 mix
HPPH
Number of his + revertant colonies
concentration (#g/plate)
TA100
TA1535
TA98
TA1537
TA1538
Saline
0 25 250
137_+9 134_+1 127_+2
13.3_+0.7 11.0_+0.5 12.0_+1.7
22.3_+3.3 31.5_+4.4 29.0_+1.1
10.0_+1.0 11.2_+1.4 8.9_+1.0
11.3_+0.7 13.1_+1.6 10.9_+2.0
Corn oil
0 25 250
132_+6 139_+4 121_+8
10.4_+2.6 14.7±1.3 13.2±0.8
21.3_+0.6 27.2_+3.1 31.0_+0.6
9.4_+1.2 13.0±1.4 9.0+_0.3
12.2_+0.9 14.1_+2.0 11.1_+0.3
PB
0 25 250
138_+7 131_+9 112_+5
14.6±1.3 12.7_+0.9 9.2-+0.2
24.0±3.0 30.1_+1.7 22.5_+3.2
7.6±1.3 9.1_+1.4 10.0_+0.5
15.1±1.8 12.6_+1.0 13.5_+2.6
BNF
0 25 126 -+ 1
141 _+ 9 133_+4 8.5 -+ 0.8
15.7 _+ 0.6 15.5_+1.8 40.5 _+ 0.3
21.7 + 0.4 45.1_+1.6 a 10.5 _+ 0.3
8.5 + 1.3 11.8-+1.2 23.5 _+ 0.8 a
10.9 -+ 2.0 31.4 -+ 1.7 a
0 25 132-+2
138_+4 137+_7 13.5-+1.4
11.7_+0.6 9.5_+0.8 46.5_+2.6 a
19.4_+3.2 41.5+_3.8 a 11.5_+0.8
7.9_+1.8 11.1-+1.9 21.0_+0.5
12.6 _ 0.9 31.5 + 0.2 a
0 25 250
123_+9 134 _+ 7 128 _+ 8
13.0_+0.5 9.6 ± 0.3 10.3 -+ 2.1
24.2_+3.1 54.0 _+ 1.4 a 46.3 _+ 2.5
7.6_+0.5 10.3 +_ 0.3 8.6 _+ 0.3
12.0 -+ 1.7 34.0 _+ 0.5 a 31.5 +_ 1.8
250 3-MC 250 PCB
Each value is the mean -+ S.E. of 5 determinations. a Only values significantly different (p < 0.01) and at least double the number of spontaneous revertants
are considered significant for mutagenicity.
Discussion Arene oxide and epoxide derivatives of several exogenous compounds, especially polycyclic aromatic hydrocarbons, have mutagenic activity. DPH biotransformation to HPPH has been suggested as proceeding through the formation of an electrophilic arene oxide. The identification, in vivo, of the 3,4-dihydro-3,4-dihydroxyphenyl metabolite of DPH (Chang et al., 1970), and data from our laboratory on the capacity of DPH to bind covalently to rat-liver microsomal proteins (Pantarotto et al., 1981), strongly support this hypothesis. In the present study, using the Ames test, we found a minor mutagenic effect of
225 TABLE 5 METABOLISM OF DPH BY $9 MIX FROM THE LIVERS OF CONTROL RATS AND RATS PRETREATED WITH DIFFERENT CYTOCHROME P.450/448 INDUCERS Treatment
DPH concentration (~g/plate)
HPPH concentration ~g/plate)
Saline
25 250 500
0.61 _+0.07 0.36 _+0.09 < 0.05
Corn oil
25 250 500
0.59 _+0.09 0.44 _+0.07 < 0.05
PB
25 250 500
0.54 _+0.13 0.50 _+0.03 < 0.05
BNF
25 250 500
1.03 + 0.11 a 0.85 _+0.06 0.21 _+0.04
3-MC
25 250 500
1.54 _ 0.10a 0.91 _+0.04 0.45 _+0.05
PCB
25 250 500
1.87 + 0.21a 1.46 _+0.09 0.86 _+0.09
Each value is the mean _+S.E. of 5 determinations. DHP, when present, was always below the limit of sensitivity of our mass-fragmentographic procedure (0.05/zg/plate). ap ~ 0.01 compared with HPPH concentrations obtained by $9 mix from the livers of corn-oil-pretreated rats.
D P H in c o n d i t i o n s o f m e t a b o l i c a c t i v a t i o n with the $9 mix f r o m the liver o f rats treated with 3-MC a n d P C B . The p r o d u c t o f D P H p - h y d r o x y l a t i o n was also f o u n d to be a slight f r a m e s h i f t m u t a g e n , a n effect observed only after H P P H i n c u b a t i o n in the presence o f $9 mix f r o m a n i m a l s pretreated with BNF, 3-MC a n d PCB. The degree o f H P P H m u t a g e n i c i t y was m o r e significant t h a n with D P H , a n d a p p e a r e d interesting in view o f recent findings f r o m o u r l a b o r a t o r y o n the m e c h a n i s m o f D H P f o r m a t i o n ( A r b o i x a n d P a n t a r o t t o , 1979). We f o u n d that D P H is t r a n s f o r m e d to D H P t h r o u g h 2 successive h y d r o x y l a t i o n s b o t h i n v o l v i n g the f o r m a t i o n of reactive species. These d a t a have recently b e e n s u p p o r t e d by the w o r k o f Lho~st et al. (1980) who identified a n epoxide-ol m e t a b o l i t e o f D P H in the u r i n e of rats treated with this drug.
226 TABLE 6 METABOLISM OF HPPH BY $9 MIX FROM THE LIVERS OF CONTROL RATS AND RATS PRETREATED WITH DIFFERENT CYTOCHROME P-450/448 INDUCERS Treatment
HPPH concentration ~g/plate)
DHP concentration (~g/plate)
Saline
25 250 500
0.27 + 0.03 0.21 +_0.02 < 0.05
Corn oil
25 250 500
0.29 _+0.05 0.19 + 0.03 < 0.05
PB
25 250 500
0.25 _+0.02 < 0.05 < 0.05
BNF
25 250 500
0.36 +_0.01 0.25 + 0.03 < 0.05
3-MC
25 250 500
0.40 + 0.04a 0.31 + 0.03 < 0.05
PCB
25 250 500
0.53 + 0.01 a 0.49 _+0.06 0.11 _+0.05
Each value is the mean _+S.E. of 5 determinations. ap < 0.01 compared with DPH concentrations obtained by $9 mix from the livers of corn-oil-pretreated rats.
It is surprising, if D P H a n d H P P H h y d r o x y l a t i o n to H P P H a n d D H P proceeds t h r o u g h the i n t e r m e d i a c y o f chemically reactive species, that a greater m u t a g e n i c effect is observed with H P P H w h e n D P H is the c o m p o u n d that is m o r e extensively h y d r o x y l a t e d in vitro. O n e e x p l a n a t i o n m i g h t lie in the half-life o f these m e t a b o l i c intermediates. T h e highly reactive arene oxide m a y well isomerize before it has e n o u g h time to reach a cell structure i n v o l v e d in the process o f m u t a t i o n . Conversely, the c h a r a c t e r i z a t i o n o f the epoxide-ol m e t a b o l i t e o f D P H in vivo suggests that this species is chemically relatively stable; hence its possible role in the H P P H m u t a g e n i c i t y seen after m e t a b o l i c activation. Parallel q u a n t i t a t i v e e v a l u a t i o n , in vitro, o f the D P H a n d H P P H h y d r o x y l a t i o n processes proved h e l p f u l in investigating the enzymes that catalyze the f o r m a t i o n o f the m u t a g e n i c m e t a b o l i c intermediates of these c o m p o u n d s . T h e c h a r a c t e r i z a t i o n o f
227
MONOOXYGENASE
•
N-H 0
I
I
H
H
DPH
OH
OH
OH
N-H
4 ISOMERIZATION
N-H
-H
0
0
I
I
H
H
DHP
MONOOXYGE NASE
0 I
H
HPPH
Fig. 1. Hypothesized mechanism of DPH metabolic conversion to HPPH and DHP.
this enzymatic system, a cytochrome-P-448-dependent mono-oxygenase, enabled us to optimize the experimental conditions so as to show up mutagenicity better. From the metabolic data reported it is evident that the mutagenic effect cannot be increased, in the case o f D P H and H P P H , by raising the concentration o f the compound being tested because of a substrate-inhibitory effect on the activating metabolizing enzymes. However, before extrapolating any definitive conclusion on D P H mutagenicity, if we assume that H P P H and D H P both represent the products o f chemical rearrangements o f reactive intermediates (see Fig. I), we must also consider that D P H and H P P H hydroxylations proceed to a much greater extent in vivo than in vitro (Pantarotto et al., 1981; Chang et al., 1972; Glazko et al., 1969). Therefore, studies are in progress in our laboratory to verify the chemical reactivity o f the new epoxide-ol D P H metabolite towards cellular macromolecules and to complete our investigations on the mutagenicity o f D P H and its metabolites in assays in vivo under conditions o f controlled biotransformation.
228
References Ames, B.N., F.D. Lee and W.E. Durston (1973) An improved bacterial test system for the detection and classification of mitogens and carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 70, 782-786. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31,347-364. Arboix, M., and C. Pantarotto (1979) Formation of electrophilic intermediates in diphenylhydantoin metabolism: Possible implications in the toxicity of this drug, 39th International Congress of Pharmaceutical Sciences, Abstract No. 100, Brighton, U.K., 3-7 Sep. Arboix, M., and C. Pantarotto (1981) Determination of 5,5-diphenylhydantoin and its major metabolites in biological specimens by gas chromatography and selected ion monitoring, J. Chromatogr., in press. Chang, T., A. Savory and A.J. Glazko (1970) A new metabolite of 5,5-diphenylhydantoin (Dilantin), Biochem. Biophys. Res. Commun., 38, 444-449. Chang, T., R.A. Okerholm and A.J. Glazko (1972) A 3-O-methylated catechol metabolite in diphenylhydantoin (dilatin) in rat urine, Res. Commun. Chem. Pathol. Pharmacol., 4, 13-23. Dam, M. (1972) Diphenylhydantoin, Neurologic aspects of toxicity, in: D.M. Woodbury, J.K. Penry and R.P. Schmidt (Eds.), Antiepileptic Drugs, Raven, New York, pp. 227-235. De Vore, G.R., and D.M. Woodbury (1977) Phenytoin: An evaluation of several potential teratogenic mechanisms, Epilepsia, 18, 387-396. Dukes, M.N.G. (1980) Anticonvulsants, in: M.N.Go Dukes (Ed.), Meyler's Side Effects of Drugs, Excerpta Medica, Amsterdam, pp. 90-101. Editorial (1971) Is phenytoin carcinogenic? Lancet, 2, 1071-1072. Glaser, G.H. (1972) Diphenylhydantoin, Toxicity, in: D.M. Woodbury, J.K. Penry and R.P. Schmidt (Eds.), Antiepileptic Drugs, Raven, New York, pp. 219-226. Glazko, A.J. (1973) Diphenylhydantoin metabolism, A prospective review, Drug Metab. Dispos., 1, 711-714. Glazko, A.J., T. Chang, J. Baukema, W.A. Dill, J.R. Goulet and R.A. Buchanan (1969) Metabolic disposition of diphenylhydantoin in normal human subjects following intravenous administration, Clin. Pharmacol. Ther., 10, 498-504. Johannessen, S.I., P.L. Morselli, C.E. Pippenger, A. Richens, D. Schmidt and H. Meinardi (Eds.) 0980) Antiepileptic Therapy: Advances in Drug Monitoring, Raven, New York. Klein, U., M. Klein, H. Sturm, M. Rothenbuhler, R. Huber, P. Stuchi, I. Gihalov, M. Heller and R. Hoigne (1976) The frequency of adverse drug reactions as dependent upon age, sex and duration of hospitalization, Int. J. Clin. Pharmacol., 13, 187-195. Kruger, G.R.F., and D. Harris (1972) Is phenytoin carcinogenic? Lancet, l, 323. Laidlaw, J., and A. Richens (Eds.) (1976) A Textbook of Epilepsy, Churchill, Edinburgh. LhoEst, G., J.M. Poupaert and M. Claesen (1980) A new metabolite of 5,5-diphenylhydantoin containing an epoxide-ol moiety, Eur. J. Mass Spectrom. Biochem. Med. Environ. Res., 1, 57-59. Loughman, P.M., H. Gold and J.C. Vance (1973) Phenytoin teratogenicity in man, Lancet, 1, 70-72. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall ( 1951) Protein measurement with the folin phenol reagent, J. Biol. Chem., 193,265-275. Pantarotto, C., M. Arboix, P. Sezzano and R. Abbruzzi (1981) Studies on 5,5-dipbenylhydantoin irreversible binding to rat liver microsomal proteins, Biochem. Pharmacol., in press.
Mutation Research,
219
Mutagenicity of diphenylhydantoin and some of its metabolites towards Salmonella typhimurium strains P. Sezzano b, A. Raimondi b, M. Arboix c and C. P a n t a r o t t o a,* a istituto di Ricerche Farmacologiche "Mario Negri" Via Eritrea 62, 20157 Milan (Italy), a lstituto di Microbiologia, Universit~ di Milano, Via Mangiagalli 31, 20133-Milan (Italy) and c Departamento de Farmacologia, Universidad Autonoma de Barcelona, Bellaterra, Barcelona (Spain)
(Accepted 3 August 1981)
Summary The mutagenicities of 5,5-diphenylhydantoin (DPH) and its major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin(HPPH) were tested in vitro using different Salmonella strains (TA1535, TA100, TA1537, TA1538, TA98). Experiments were carried out at various concentrations in the absence and in the presence of an activating system consisting of hepatic $9 fraction from control rats and from rats pretreated with phenobarbital (PB), /3-naphthoflavone (BNF), 3-methylcholanthrene (3-MC) and Aroclor 1254 (PCB). DPH slightly increased the number of revertants per plate only after incubations with TA1538 in the presence of the $9 fraction from the liver of 3-MC- and PCBpretreated animals. A similar but more significant frameshift mutation was observed for HPPH on both TA98 and TA1538 strains and in conditions of metabolic activation by the liver microsomal fractions of rats after pretreatment with BNF, 3-MC and especially PCB. Parallel experiments on the metabolism of DPH to HPPH and of HPPH to the catechol derivative in vitro support the hypothesis of an involvement of epoxide intermediates in the mutagenic activity of DPH.
5,5-Diphenylhydantoin (DPH) is widely used in clinical practice for the treatment of epilepsy (Laidlaw and Richens, 1976; Johannessen et al., 1980). Neurological, hematological, immunological and hepatic toxicity in subjects under therapy with DPH has been reported by several authors (Glaser, 1972; Dam, 1972; Klein et al., 1976; Dukes, 1980), and the possibility of this drug having teratogenic and carci*To whomcorrespondenceshould be addressed. 0165-7992/82/0000-0000/$02.75 © ElsevierBiomedicalPress
220 nogenic effects has also been discussed (Loughman et al., 1973; De Vore and Woodbury, 1977; Editorial, 1971; Kruger and Harris, 1972). Some of these toxicities might be related to DPH metabolism through the formation of chemically reactive intermediates. Reports in the literature show that DPH is metabolized to phenolic, dihydrodiol and catechol derivatives and discuss the possible intermediacy of epoxides (Glazko, 1973). This hypothesis has been supported by recent results from our group on irreversible DPH binding to rat-liver microsomal proteins catalyzed by a mono-oxygenase dependent on cytochrome P-448 (Pantarotto et al., 1981). It is known that epoxide intermediates produced during the biotransformation of exogenous compounds are responsible for their mutagenic effects. Therefore it seemed interesting to us to study the mutagenicity of DPH and HPPH in vitro and in conditions of controlled metabolism.
Materials and methods
Chemicals. DPH, HPPH and 5-(4-methylphenyl)-5-phenylhydantoin (MPPH) were obtained from Aldrich, Beerse (Belgium). 5-(3,4-Dihydroxyphenyl)-5-phenylhydantoin (DHP), as reference material, was extracted with chloroform at pH 4.5 from the urine of DPH-treated rats and first purified by preparative thin-layer chromatography on silica-gel glass plates developed in a solvent system consisting of chloroform : methanol : acetic acid, 90:10 : 1 (v/v). The Rf value of DHP was 0.37. Further purification was achieved by high-performance liquid chromatography on a reversed-phase column (0.25 m long × 2.6 mm i.d.) packed with 10 #m (average particle diameter) LiChrosorb RP-18 (Merck, Darmstadt, West-Germany). The column was eluted with a mixture of acetonitrile and acetate buffer, 0.02 M, pH 4.5 (40: 60, v/v). PB was obtained from Merck, Darmstadt (West-Germany); 3-MC from Sigma, St. Louis, MO (U.S.A.); BNF from Aldrich, Beerse (Belgium); and PCB from Analabs, North Haven, CN (U.S.A.). All reagents were analytical grade. Animals. Male CD-COBS rats (200-220 g, b.w.) from Charles River Italy (Calco, Como, Italy) were used. Hepatic microsomal cytochrome P-450/448 was induced with PB (40 mg/kg i.p., in 0.9% saline, twice daily for 3 days), BNF (60 mg/kg i.p., in corn oil, once daily for 2 days), 3-MC (40 mg/kg i.p., in corn oil, once daily for 3 days) or PCB (500 mg/kg i.p., in corn oil, once). All rats were fasted for 24 h before they were killed. Preparation o f liver 9000 × g supernatant fractions. Groups of 5 pretreated rats were used for the preparation of each batch of liver 9000 × g supernatant fractions ($9). Control rats and rats pretreated with PB, BNF and 3-MC were killed by decapitation 24 h after the last treatment, and PCB-pretreated animals were killed after 5 days. The livers were excised and pooled, and $9 fractions were obtained
221
according to the procedure of Ames et al. (1975). Proteins were determined by the method of Lowry et al. (1951) with albumin fraction V as the standard. Mutagenicity assay. Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 were used. The mutagenicity test was carried out in airtight petri dishes according to the method of Ames et al. (1973). DPH and H P P H were dissolved in dimethyl sulfoxide (DMSO) and added at 45 °C to 2 ml of molten top agar with added L-histidine. HC1 (0.5 mM) and biotin (0.5 mM) together with 0.1 ml of an overnight nutrient broth culture of the bacterial tester strains and, if required, 0.5 ml of $9 fraction fortified with an NADPH-regenerating system ($9 mix). Samples were plated on Vogel-Bonner minimal medium dishes and incubated at 37 °C for 48 h. After incubation, the number of histidine-revertant colonies was determined.
Gas chromatographic-mass fragmentographic determination of HPPH and DHP H P P H and DHP were determined according to Arboix and Pantarotto (1981). The incubation medium was resuspended in 6 ml of a 0.3 M NaH2PO4.H20 aqueous solution containing 500 ng of MPPH as internal standard. Hermetically sealable glass tubes bearing a Teflon disk in the inner part of the cap were used, and the samples were extracted with 5 ml of 0.5 M trideuteromethyl iodide methylene chloride solution after addition of 0.5 ml of 10 N NaOH and 100 /~1 of 0.1 M tetrahexylammonium hydrogen sulfate solution in 0.1 N NaOH. Extractions were carried out for 2 h by shaking at 60 °C in subdued light. The organic phase (4.5 ml) was transferred to conical glass tubes and evaporated to dryness under a gentle stream of nitrogen in a water-bath at 35 °C. The residue was resuspended in 50/~1 of methylene chloride, and the tetrahexylammonium salt was precipitated by addition of 2 ml of n-hexane. The tubes were capped and centrifuged for 5 min at 4000 x g. The supernatant fraction (1.8 ml) was transferred to conical glass tubes and evaporated to dryness under a nitrogen stream at 35 °C. The residue was dissolved in 100 #1 of Tri-Deuter-8® solution (Pierce, Rockford, IL, U.S.A.), and a 1 - 5 / d portion of this solution was injected for mass-fragmentographic analysis. Tri-Deuter-8® was used to achieve complete derivatization of DHP. For mass fragmentography the instrument (LKB 2091 mass spectrometer equipped with a computer system model 2130 for data acquisition and calculation) was focused on the ions at m/e 300 (molecular ion in the spectrum of MPPH ditrideuteromethyl derivative), 319 (molecular ion in the spectrum of H P P H tritrideuteromethyl derivative) and 352 (molecular ion in the spectrum of DHP tetratrideuteromethyl derivative).
Results
DPH was tested for mutagenicity in various Salmonella typhimurium strains
222 TABLE 1 MUTAGENICITY OF DPH FOR 5 SALMONELLA STRAINS DHP concentration ~g/plate) 0 1 10 l~ 1000
Number of His÷ revertant colonies TA100
TA1535
TA98
TA1537
TA1538
136± 8 133± 6 124±10 130± 4 136± 9
16.0±1.9 10.6±1.4 16.2±1.8 17.0±0.5 14.5±1.8
~.2±2.2 28.0±2.3 21.2±2.8 23.3±1.7 20.5±3.7
6.7±1.2 5.6±0.6 8.0±1.0 7.0±2.0 8.3±2.0
13.5±1.6 17.0±2.3 14.7±1.7 11.9±2.5 13.5±1.0
Each value is the mean ± S,E. of 5 determinations.
(TA100, T A 1 5 3 5 , T A 9 8 , T A 1 5 3 7 , TA1538), a n d the results o f these e x p e r i m e n t s are r e p o r t e d in T a b l e 1. T h e d r u g was n o t m u t a g e n i c at a n y o f the c o n c e n t r a t i o n s tested, r a n g i n g f r o m 1 to 1000/~g/plate. S i m i l a r results were o b t a i n e d with H P P H at the s a m e c o n c e n t r a t i o n s ( T a b l e 2). I n b o t h e x p e r i m e n t s , cell survival was 100°70 indic a t i n g t h a t the d r u g a n d its h y d r o x y l a t e d m e t a b o l i t e were n o t c y t o t o x i c even a t doses n e a r the limit o f their solubility. T a b l e s 3 a n d 4 s h o w the m u t a g e n i c i t y o f D P H a n d H P P H w h e n i n c u b a t i o n s were c a r r i e d o u t in the presence o f $9 mix f r o m the livers o f c o n t r o l rats a n d rats pret r e a t e d with d i f f e r e n t c y t o c h r o m e P - 4 5 0 / 4 4 8 i n d u c e r s . T h e c a p a c i t y o f $9 mix p r e p a r a t i o n s to a c t i v a t e a p r o m u t a g e n was tested with b e n z o [ a ] p y r e n e at a dose o f 5 /zg/plate. W i t h $9 mix f r o m P C B - p r e t r e a t e d rats, a 7-fold i n c r e a s e in the n u m b e r o f s p o n t a n e o u s r e v e r t a n t colonies was o b s e r v e d . A f t e r m e t a b o l i c a c t i v a t i o n by the $9 mix f r o m the livers o f 3 - M C - a n d P C B - p r e t r e a t e d rats, D P H at c o n c e n t r a t i o n s , respectively, o f 25 a n d 250 # g / p l a t e , h a d limited m u t a g e n i c activity o n l y t o w a r d s T A 1 5 3 8 . M o r e significant results were o b s e r v e d for H P P H w h i c h a l s o p r o v e d to be a slight f r a m e s h i f t m u t a g e n . Statistically significnt d a t a (p < 0.01) were o b t a i n e d w h e n H P P H was tested on strains T A 9 8 a n d T A 1 5 3 8 at the r e p o r t e d c o n c e n t r a t i o n s a n d in the presence o f $9 mix f r o m rats p r e t r e a t e d with B N F , 3 - M C a n d especially
TABLE 2 MUTAGENICITY OF HPPH FOR 5 SALMONELLA STRAINS HPPH concentration ~g/plate)
Number of His + revertant colonies TA100
TA1535
TA98
TA 1537
TA1538
0 1 10 1~ 1000
140±12 128± 2 139±10 132±11 122±14
11.2±0.5 16.0±2.2 13.3±1.7 12.0±1.8 14.0±2.3
20.0±2.0 21.0±2.1 21.6±1.6 23.0±4.0 27.0±4.5
7.0±1.1 8.1±2.0 10.0±1.4 7.6±1.6 8.3±1.8
11.0±2.1 15.3±0.8 11.3±2.9 12.8±1.1 13.0±1.0
Each value is the mean ± S.E. of 5 determinations.
223
TABLE 3 MUTAGENICITY OF DPH FOR 5 SALMONELLA STRAINS IN THE PRESENCE OF $9 MIX OBTAINED FROM THE LIVERS OF CONTROL RATS AND RATS PRETREATED WITH DIFFERENT CYTOCHROME P-450/448 INDUCERS DPH concentration (~g/plate)
Number of His + revertant colonies TA100
TA1535
TA98
TA1537
TA1538
Saline
0 25 250
129± 1 134± 6 130± 2
14.3±0.6 15.0±1.4 11.0±2.1
26.1±1.8 22.3±1.3 20.0±2.4
9.0±1.1 7.2+0.8 11.0±1.3
11.0±2.1 12.3±1.6 13.6±2.3
Corn oil
0 25 250
140± 8 137±12 129± 7
13.7±0.2 9.9±2.3 15.3±1.6
26.6±0.5 21.0±1.2 27.3±2.7
10.3±1.2 8.7±0.6 7.4±1.3
13.7±0.2 17.1±0.9 15.0±1.4
PB
0 25 250
130± 5 139± 2 126± 3
8.9±2.3 13.5±0.2 11.3±1.2
19.7±3.0 25.4±0.5 21.9±1.7
8.6±1.7 6.5±0.5 7.3+2.8
12.5±2.0 11.2±1.1 14.6+2.0
BNF
0 25 250
127± 5 135± 2 140± 6
14.2±2.0 12.6±0.3 15.3±2.1
25.2±2.4 22.3±2.6 23.0±0.8
8.9±0.4 13.0±0.5 14.3±2.7
14.7+1.7 20.0±2.3 23.0+2.6
3-MC
0 25 250
120± 6 135± 1 138± 4
10.8±1.2 12.6±0.3 9.6±0.3
20.4±2.3 33.0±3.5 36.5±2.0
7.8±1.6 9.0±0.5 11.5±1.4
13.4±0.8 31.6±0.3 a 16.5±1.5
PCB
0 25 250
141± 4 141±12 137± 4
14.6±2.0 13.3±0.8 11.6±1.4
20.4±3.2 37.3±2.1 26.1±3.2
9.6±1.3 11.6±2.0 11.0±1.6
13.7±1.2 27.0+1.1 ~.8±2.6 a
$9 mix
Each value is the mean +_S.E. of 5 determinations. a Only values significantly different (p < 0.01) and at least double the number of spontaneous revertants are considered significant for mutagenicity.
P C B . A t the dose o f 1000 # g / p l a t e , D P H and H P P H m u t a g e n i c i t y was no l o n g e r detectable, p r o b a b l y because o f a substrate i n h i b i t o r y e f f e c t o n d r u g - m e t a b o l i z i n g enzymes. This h y p o t h e s i s is s u p p o r t e d by q u a n t i t a t i v e e v a l u a t i o n o f D P H an d H P P H m e t a b o l i s m , s h o w n in T a b l e s 5 a n d 6. D e t e r m i n a t i o n s w e r e car r i ed o u t in the s a m e i n c u b a t i o n m e d i a as f o r the m u t a g e n i c i t y test. Th e d a t a indicate t h a t H P P H an d D H P f o r m a t i o n f r o m D P H a n d H P P H , respectively, is c a t a l y z e d by a c y t o c h r o m e P - 4 4 8 - d e p e n d e n t m o n o - o x y g e n a s e , these h y d r o x y l a t i o n s being i n d u ced by B N F , 3M C a n d P C B b u t n o t by P B . T h e f a il u r e to d e t e r m i n e D H P af t er i n c u b a t i o n s with D P H m i g h t be related to the limit o f sensitivity o f o u r m a s s - f r a g m e n t o g r a p h i c proc e d u r e w h i c h in these c o n d i t i o n s was 50 n g / s a m p l e .
224 TABLE 4 M U T A G E N I C I T Y OF H P P H FOR 5 SALMONELLA STRAINS IN T H E PRESENCE OF $9 MIX O B T A I N E D FROM T H E LIVERS OF CONTROL RATS AND RATS PRETREATED W I T H DIFFERENT C Y T O C H R O M E P-450/448 INDUCERS $9 mix
HPPH
Number of his + revertant colonies
concentration (#g/plate)
TA100
TA1535
TA98
TA1537
TA1538
Saline
0 25 250
137_+9 134_+1 127_+2
13.3_+0.7 11.0_+0.5 12.0_+1.7
22.3_+3.3 31.5_+4.4 29.0_+1.1
10.0_+1.0 11.2_+1.4 8.9_+1.0
11.3_+0.7 13.1_+1.6 10.9_+2.0
Corn oil
0 25 250
132_+6 139_+4 121_+8
10.4_+2.6 14.7±1.3 13.2±0.8
21.3_+0.6 27.2_+3.1 31.0_+0.6
9.4_+1.2 13.0±1.4 9.0+_0.3
12.2_+0.9 14.1_+2.0 11.1_+0.3
PB
0 25 250
138_+7 131_+9 112_+5
14.6±1.3 12.7_+0.9 9.2-+0.2
24.0±3.0 30.1_+1.7 22.5_+3.2
7.6±1.3 9.1_+1.4 10.0_+0.5
15.1±1.8 12.6_+1.0 13.5_+2.6
BNF
0 25 126 -+ 1
141 _+ 9 133_+4 8.5 -+ 0.8
15.7 _+ 0.6 15.5_+1.8 40.5 _+ 0.3
21.7 + 0.4 45.1_+1.6 a 10.5 _+ 0.3
8.5 + 1.3 11.8-+1.2 23.5 _+ 0.8 a
10.9 -+ 2.0 31.4 -+ 1.7 a
0 25 132-+2
138_+4 137+_7 13.5-+1.4
11.7_+0.6 9.5_+0.8 46.5_+2.6 a
19.4_+3.2 41.5+_3.8 a 11.5_+0.8
7.9_+1.8 11.1-+1.9 21.0_+0.5
12.6 _ 0.9 31.5 + 0.2 a
0 25 250
123_+9 134 _+ 7 128 _+ 8
13.0_+0.5 9.6 ± 0.3 10.3 -+ 2.1
24.2_+3.1 54.0 _+ 1.4 a 46.3 _+ 2.5
7.6_+0.5 10.3 +_ 0.3 8.6 _+ 0.3
12.0 -+ 1.7 34.0 _+ 0.5 a 31.5 +_ 1.8
250 3-MC 250 PCB
Each value is the mean -+ S.E. of 5 determinations. a Only values significantly different (p < 0.01) and at least double the number of spontaneous revertants
are considered significant for mutagenicity.
Discussion Arene oxide and epoxide derivatives of several exogenous compounds, especially polycyclic aromatic hydrocarbons, have mutagenic activity. DPH biotransformation to HPPH has been suggested as proceeding through the formation of an electrophilic arene oxide. The identification, in vivo, of the 3,4-dihydro-3,4-dihydroxyphenyl metabolite of DPH (Chang et al., 1970), and data from our laboratory on the capacity of DPH to bind covalently to rat-liver microsomal proteins (Pantarotto et al., 1981), strongly support this hypothesis. In the present study, using the Ames test, we found a minor mutagenic effect of
225 TABLE 5 METABOLISM OF DPH BY $9 MIX FROM THE LIVERS OF CONTROL RATS AND RATS PRETREATED WITH DIFFERENT CYTOCHROME P.450/448 INDUCERS Treatment
DPH concentration (~g/plate)
HPPH concentration ~g/plate)
Saline
25 250 500
0.61 _+0.07 0.36 _+0.09 < 0.05
Corn oil
25 250 500
0.59 _+0.09 0.44 _+0.07 < 0.05
PB
25 250 500
0.54 _+0.13 0.50 _+0.03 < 0.05
BNF
25 250 500
1.03 + 0.11 a 0.85 _+0.06 0.21 _+0.04
3-MC
25 250 500
1.54 _ 0.10a 0.91 _+0.04 0.45 _+0.05
PCB
25 250 500
1.87 + 0.21a 1.46 _+0.09 0.86 _+0.09
Each value is the mean _+S.E. of 5 determinations. DHP, when present, was always below the limit of sensitivity of our mass-fragmentographic procedure (0.05/zg/plate). ap ~ 0.01 compared with HPPH concentrations obtained by $9 mix from the livers of corn-oil-pretreated rats.
D P H in c o n d i t i o n s o f m e t a b o l i c a c t i v a t i o n with the $9 mix f r o m the liver o f rats treated with 3-MC a n d P C B . The p r o d u c t o f D P H p - h y d r o x y l a t i o n was also f o u n d to be a slight f r a m e s h i f t m u t a g e n , a n effect observed only after H P P H i n c u b a t i o n in the presence o f $9 mix f r o m a n i m a l s pretreated with BNF, 3-MC a n d PCB. The degree o f H P P H m u t a g e n i c i t y was m o r e significant t h a n with D P H , a n d a p p e a r e d interesting in view o f recent findings f r o m o u r l a b o r a t o r y o n the m e c h a n i s m o f D H P f o r m a t i o n ( A r b o i x a n d P a n t a r o t t o , 1979). We f o u n d that D P H is t r a n s f o r m e d to D H P t h r o u g h 2 successive h y d r o x y l a t i o n s b o t h i n v o l v i n g the f o r m a t i o n of reactive species. These d a t a have recently b e e n s u p p o r t e d by the w o r k o f Lho~st et al. (1980) who identified a n epoxide-ol m e t a b o l i t e o f D P H in the u r i n e of rats treated with this drug.
226 TABLE 6 METABOLISM OF HPPH BY $9 MIX FROM THE LIVERS OF CONTROL RATS AND RATS PRETREATED WITH DIFFERENT CYTOCHROME P-450/448 INDUCERS Treatment
HPPH concentration ~g/plate)
DHP concentration (~g/plate)
Saline
25 250 500
0.27 + 0.03 0.21 +_0.02 < 0.05
Corn oil
25 250 500
0.29 _+0.05 0.19 + 0.03 < 0.05
PB
25 250 500
0.25 _+0.02 < 0.05 < 0.05
BNF
25 250 500
0.36 +_0.01 0.25 + 0.03 < 0.05
3-MC
25 250 500
0.40 + 0.04a 0.31 + 0.03 < 0.05
PCB
25 250 500
0.53 + 0.01 a 0.49 _+0.06 0.11 _+0.05
Each value is the mean _+S.E. of 5 determinations. ap < 0.01 compared with DPH concentrations obtained by $9 mix from the livers of corn-oil-pretreated rats.
It is surprising, if D P H a n d H P P H h y d r o x y l a t i o n to H P P H a n d D H P proceeds t h r o u g h the i n t e r m e d i a c y o f chemically reactive species, that a greater m u t a g e n i c effect is observed with H P P H w h e n D P H is the c o m p o u n d that is m o r e extensively h y d r o x y l a t e d in vitro. O n e e x p l a n a t i o n m i g h t lie in the half-life o f these m e t a b o l i c intermediates. T h e highly reactive arene oxide m a y well isomerize before it has e n o u g h time to reach a cell structure i n v o l v e d in the process o f m u t a t i o n . Conversely, the c h a r a c t e r i z a t i o n o f the epoxide-ol m e t a b o l i t e o f D P H in vivo suggests that this species is chemically relatively stable; hence its possible role in the H P P H m u t a g e n i c i t y seen after m e t a b o l i c activation. Parallel q u a n t i t a t i v e e v a l u a t i o n , in vitro, o f the D P H a n d H P P H h y d r o x y l a t i o n processes proved h e l p f u l in investigating the enzymes that catalyze the f o r m a t i o n o f the m u t a g e n i c m e t a b o l i c intermediates of these c o m p o u n d s . T h e c h a r a c t e r i z a t i o n o f
227
MONOOXYGENASE
•
N-H 0
I
I
H
H
DPH
OH
OH
OH
N-H
4 ISOMERIZATION
N-H
-H
0
0
I
I
H
H
DHP
MONOOXYGE NASE
0 I
H
HPPH
Fig. 1. Hypothesized mechanism of DPH metabolic conversion to HPPH and DHP.
this enzymatic system, a cytochrome-P-448-dependent mono-oxygenase, enabled us to optimize the experimental conditions so as to show up mutagenicity better. From the metabolic data reported it is evident that the mutagenic effect cannot be increased, in the case o f D P H and H P P H , by raising the concentration o f the compound being tested because of a substrate-inhibitory effect on the activating metabolizing enzymes. However, before extrapolating any definitive conclusion on D P H mutagenicity, if we assume that H P P H and D H P both represent the products o f chemical rearrangements o f reactive intermediates (see Fig. I), we must also consider that D P H and H P P H hydroxylations proceed to a much greater extent in vivo than in vitro (Pantarotto et al., 1981; Chang et al., 1972; Glazko et al., 1969). Therefore, studies are in progress in our laboratory to verify the chemical reactivity o f the new epoxide-ol D P H metabolite towards cellular macromolecules and to complete our investigations on the mutagenicity o f D P H and its metabolites in assays in vivo under conditions o f controlled biotransformation.
228
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