Mutation Research, 129 (1984) 291-297 291 Elsevier

MTR 03947

NADPH-generating system: influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and

mouse $9 fractions *

Giorgio Cante|li Forti ~, Moreno Paolini ~, Patrizia Hrelia ~, Claudio Corsi 2, Gian Luigi Biagi 1 and Giorgio Bronzetti 2

I lstituto di Farmacologia dell'Universit~ Via Irnerio, 48, 40126 Bologna (Italy) and 2 Istituto di Mutagenesi e Differenziamento, C.N.R., Via Svezia, 10, 56100 Pisa (Italy)

(Received 27 December 1983) (Revision received 18 July 1984)

(Accepted 23 July 1984)

Summary

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroani- sole O-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with $9 liver fractions from mice (induced with Na-phenobarbital and fl-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D 7 strain of Saccharomyces cerevisiae.

This work was presented at the 13th Annual Meeting of the European Environmental Mutagen Society, 5-9 September 1983, Montpellier (France). Correspondence to: Prof. Giorgio Cantelli Forti, lstituto di Farmacologia, Via Irnerio, 48, 40126 Bologna (Italy).

Abbreviations: APD, aminopyrine demethylase; p-NAD, p- nitroanisole demethylase; PB, sodium phenobarbital; fl-NF, fl-naphthoflavone; G-6-P, glucose 6-phosphate; G-6-PDH, glucose-6-phosphate dehydrogenase; LMA, liver-microsomal assay; DMSO, dimetbyl sulphoxide; ED, 7-ethoxycoumarin deethylase; DMN, dimethylnitrosamine; AR2MNFN, a nit roimidazo[2,1 -b]thiazole derivative.

Many chemicals require metabolic activation before they can react with DNA and exert muta- genic and/or carcinogenic effects (Miller and Miller, 1977; Oesch, 1979).

NADPH and molecular oxygen are known to be required by the hepatic microsomal cytochrome P-450 mono-oxygenase activity system of the post-mitochondrial supernatant ($9 fraction) for bioactivation of precarcinogens and premutagens in the genotoxicity test. However, complex bio-

0027-5107/84/$03.00 © 1984 Elsevier Science Publishers B.V.

292

chemical phenomena occur in incubation mixtures for microsomal assays and, particularly the pres- ence of some xenobiotics (Paolini et al., 1983) can affect various phase I and phase II enzyme activi- ties of the drug metabolizing system. So, the bal- ance of activation/inactivation pathways and the mutagenic response can change. In fact the differ- ent denaturing and/or inhibitory effects act dif- ferentially towards the components of the NADPH-generating system: inactivating agents (such as proteolytic enzymes, active metabolic in- termediates, lipid peroxidation products, etc.) may act on G-6-PDH and/or cytosolic enzymes (such as nucleotide pyrophosphatase, glycohydrolase, phosphorylase, oxidase, etc.) and may destroy the NADPH itself. In the normal conditions for LMA, the amount of NADPH is probably inadequate to produce optimal mono-oxygenase activity, particu- larly after a long period of time. Thurman and Scholz (1969), for example, have demonstrated in perfused rat liver that the generation of NADPH may limit the demethylation of aminopyrine under certain conditions.

These considerations indicate that studies for in vitro drug metabolism may alter the steady-state levels of NADPH.

The metabolic activating system ($9 fraction) as generally applied utilizes endogenous G-6-PDH by adding ofily Mg 2÷, glucose 6-phosphate (G-6-P) and NADP + for the NADPH-generating system (Zimmermann et al., 1975; Ames et al., 1975).

Only few authors occasionally added NADPH and G-6-PDH (see for example Loprieno et al., 1976; Bronzetti et al., 1981) or, as suggested by Yahagi et al. (1975), NADH to the microsomal fraction (see for example Yahagi et al., 1978; Prival et al., 1982; Mori et al., 1983).

In order to establish the role of the NADPH-re- generating system on the behaviour of the mono- oxygenase, the present work evaluates the stability of ED, APD and p-NAD activities with and without exogenous G-6-PDH in the incubation mixtures for the LMA. The stability of G-6-PDH was also determined.

Finally, we studied the influence of exogenous G-6-PDH in the incubation mixtures for LMA on the sensitivity of the test for mutagenicity, using dimethylnitrosamine (DMN) and AR2MNFN as premutagens.

Materials and Methods

Chemicals Aroclor 1254 was a generous gift from Prof.

Marvin S. Legator (University of Texas, Medical Branch at Galveston, U.S.A.). Dimethylnitrosa- mine was obtained from Schuchardt Co., Munich. The 7-ethoxycoumarin, NADP + and glucose 6- phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). Glucose-6-phosphate dehy- drogenase was purchased from Boehringer (Mann- heim, F.R.G.). AR2MNFN was synthesized in our laboratory (Fig. 1). Spectrograde dimethylsulpho- xide (DMSO) was purchased from Farmitalia- Carlo Erba (Milan, Italy).

Yeast strain Saccharomyces cerevisiae strain D7, obtained

from Prof. F.K. Zimmermann, was used to mea- sure the frequency of mitotic gene conversion at the trp locus and mitotic recombination between the centromere and the ade2 locus (Zimmermann et al., 1975; Bronzetti et al., 1978 and 1981).

Animal induction and preparation of $9 fraction Female Swiss Albino mice, CD1 strain and

female Sprague-Dawley rats, maintained on a standard diet, were used for the preparation of the $9 hepatic fraction. The mice received Na-pheno- barbital (100 mg/kg) i.p. the first day, Na-pheno- barbital (50 mg/kg) and fl-naphthoflavone (80 mg/kg) i.p. the second day (Matsushima et al., 1976) and Na-phenobarbital (100 mg/kg) i.p. the third day. They were killed on the fourth day. The rats were induced with Aroclor 1254 in a single i.p. injection of 500 mg/kg 5 days before sacrifice as described by Ames et al. (1975). The animals were killed by cervical dislocation after stunning by rotation; the livers were removed aseptically and homogenized, at 4 ml/g, in 0.01 M N a + / K +

F~--~CH 3

Fig. 1. Structure of the nitroimidazo[2,1-h]thiazole derivativ~ AR2MNFN.

293

phosphate buffer, pH 7.4, containing KC1 1.15% (w/v), and the $9 fractions were prepared as reported before (Bronzetti et al., 1981).

cells/105 and 103 survivors, respectively, and on complete medium for survivor counts (Zimmer- mann et al., 1975; Bronzetti et al., 1978, 1981).

Metabolizing enzymes 7-Ethoxycoumarin O-deethylase activity was

determined by the method of Aitio (1978). The aminopyrine N-demethylase activity (APD) was determined by quantitating the formaldehyde re- lease by the Nash reagent, essentially according to Mazel (1971) with some modifications to obtain linearity of the assay response with our enzyme preparation. The determination of p-nitroanisole O-demethylase activity (p-NAD) was similar to APD, the substrate being a nearly saturated solu- tion of p-nitroanisole (Zannoni et al., 1971). The enzyme stability was determined in the conditions of LMA at time 0, after 1 and 2 h of pre-incuba- tion in the presence of 1 mM NADP ÷, 6 mM G-6-P, 4 mM MgC12, and 1 ml of $9 in a total volume of 4 ml, obtained with 3 ml of 0.1 M N a + / K ÷ phosphate buffer, pH 7.4, with and without 20 /tl of G-6-PDH (microcrystalline sus- pension: grade II) added in the incubation mix- tures (final concentration 0.70 U/ml) .

The statistical analysis was made by the rank method of Wilcoxon on data separately obtained on groups of 5 animals.

Determination of glucose-6-phosphate dehydro- genase and protein concentration

The glucose-6-phosphate dehydrogenase was as- sayed using the procedure of Boehringer-Man- nheim (1975) and the protein concentration was determined according to Lowry (1951) as reported by Bailey (1967).

Suspension test procedure The yeast cells in stationary phase (about 6 ×

108 cells) were incubated in the liver-microsomal assay in the presence of various concentrations of DMN (final concentration 50, 100 and 200 mM) and AR2MNFN (final concentration 0.5, 5 × 10 -2 and 5 × 10 -3 /~M). The experiments were per- formed with and without exogenous G-6:PDH (final concentration 0.7 U/ml) . The mixtures were incubated for 2 h at 37°C. Then they were plated on selective media to evaluate trp + convertants and ade- recombinants expressed as number of

Results

Figs. 2-4 report the stability curves for ED, APD and p-NAD, respectively, obtained over a period of 2 h with and without G-6-PDH in the incubation mixtures, containing liver $9 fractions from mouse or rat.

In the presence of exogenous G-6-PDH, the p-NAD activity was more stable than the APD activity, in the mouse. In contrast, in the rat the APD activity was more stable. The ED activity was less stable than APD and p-NAD activities in

.! e~

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1.o[ I 0.5

i

Time of pre-incubation (hours)

4.o ] "7 MOUSE

3.0

~ 2.0 7

1.0

I 2 Time of pre-incubation (hours)

Fig. 2. Behaviour of 7-ethoxycoumarin O-deethylase (ED) dur- ing incubations for liver-microsomal assay, with (m) and without (A) G-6-PDH. Each point represents the mean___S.D, of 5 Expts.

7

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.-4 {

'rime of pre-incubation (hoursj

I

MOUSE 9

i 7

5

×

= 3

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294

1 2 'rime of pee-incubation (hours}

Fig. 3. Bchaviour of aminopyrine N-demethylase (APD) during incubations for l iver-microsomal assay, with (m) and without

(*,) G-6-PDH. Each point represents the mean+S.D, of 5 Expts.

both species investigated. In the mouse, the ab- sence of exogenous G-6-PDH produces a signifi- cant loss ( p < 0.01) of the studied activities at 0 time. At the subsequent times of determination it is possible to observe an increase in stability for all the parameters, except for p - N A D for which sta- bility is reduced.

In the rat, only ED had a similar behaviour, while the other enzyme stabilities showed a signifi- cant decrease after 2 h of incubation ( p < 0.01 and p < 0.02 for A P D and p - N A D respectively).

The endogenous G-6-PDH activity in liver $9 fractions from mouse and rat were also de- termined. Fig. 5 shows that this activity is signifi- cantly lower ( p < 0.01) in the mouse than in the rat at 0 time; however, the behaviour of G-6-PDH was the same in both species considered, as judged

7

0

7 e-

al

I

" ' - ' t

0 1 Time of pPe-incubation (hours)

? 3

7 • 5 z

I

0 1 2 l'ime of pre-inctlbat ion (hours)

Fig. 4. Behaviour of p-nitroanisole O-demethylase (p-NAD) during incubations for liver-microsomal assay, with (I) and without (A) G-6-PDH. Each point represents the mean ! S.D. of 5 Expts.

I 0 . 4 ~

=0.34

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0 1 2 Time of prc-incubat.ion {hours)

Fig. 5. Behaviour of glucose-6-phosphate dehydrogena~ ((}-6- PDH) during incubations for l iver-microsomal assay. Each point represents the mean_# S.D, o[ 5 Expts,

295

TABLE 1

INDUCTION OF MITOTIC GENE CONVERSION AND MITOTIC CROSSING-OVER IN Saccharomyces cerevisiae STRAIN D 7 BY DMN AND AR2MNFN IN THE SUSPENSION TEST WITH METABOLIC ACTIVATION +_ G-6-PDH a

Compound Conc. Survival (%) ade- recombinants/103 surv. trp + convertants/105 surv.

- G-6-PDH + G-6-PDH - G-6-PDH + G-6-PDH - G-6-PDH + G-6-PDH

Control mice

DMN

AR2MNFN

Control rat

DMN

AR2MNFN

100 (2752) 100 (2014) 0.30 (1) 0.40 (1) 0.44 (121) 0.98 (197)

50 mM 100 (2 763) 100 (2191) 0.74 (2) 3.91 (9) 0.87 (240) 1.52 (333) 100 mM 88 (2414) 99 (1989) 2.16 (5) 4.31 (6) 0.97 (234) 3.04 (605) 200 mM 75 (2050) 98 (1 977) 2.38 (5) 4.34 (9) 1.02 (209) 4.43 (876)

5 x 10-3/tM 87 (2 387) 91 (1 840) 1.07 (9) 3.27 (6) 0.76 (181) 3.19 (587) 5x10 Z~M 79(2182) 89(1787) 1.45(3) 3.60 (6) 0.84(183) 3.32(593) 0.5 ~M 78 (2159) 81 (1 640) 3.19 (7) 3168 (6) 1.07 (231) 4.07 (667)

- 100 (3029) 100 (3 368) 1.16 (4) 1.70 (6) 0.51 (154) 0.95 (320)

50 mM 81 (2468) 81 (2733) 1.17 (3) 2.16 (6) 0.67 (165) 1.05 (287) 100 mM 81 (2463) 74 (2605) 2.04 (5) 8.60 (22) 1,02 (216) 2.70 (676) 200 mM 70 (2134) 61 (2070) 3.26 (7) 10.08 (21) 154 (329) 3.98 (824)

5x10 3 /~M 91(2759) 82(2770) 2.24(6) 4.82(13) 1.07(295) 1.94(537) 5×10 2 /~M 89(2697) 76(2550) 2.91(8) 7.97(20) 1.44(388) 2.34(597) 0.5 ~tM 84 (2558) 69 (2 310) 3.04 (8) 8.94 (21) 1.75 (448) 2.51 (580)

a In parentheses the colonies counted. The results are the media of 3-4 Expts.

f rom the percentage falls evaluated after 1 and 2 h of incuba t ion for LMA. In par t icular , af ter 2 h the fall was 57.1% + 8.1 and 52.8% + 17.4 for mouse and rat, respectively.

Table 1 shows the genetic effects (mitot ic gene convers ion and mi to t ic crossing-over) ob ta ined on

the 0 7 s train of Saccharomyces cerevisiae in the presence and absence of exogenous G - 6 - P D H with

the test compounds . The results indicate that the add i t ion of G - 6 - P D H in the incuba t ion mixtures enhanced the genetic response.

Discussion

The zero t ime values, for the various phase I enzyme activit ies invest igated, indicate that endog- enous G - 6 - P D H (or the regenera ted N A D P H ) is no t sufficient to supply all the specific i soenzymes (Guenger ich et al., 1982) of microsomal mono- oxygenase activit ies studied, in the mouse. On the o ther hand, the slight increase of s tabi l i ty dur ing the incuba t ion for L M A in the absence of G-6-

PDH, does not make up for the marked ly lower act ivi ty at t ime 0.

In the rat, on the other hand, the endogenous

N A D P H supply appears to be sufficient, at least for A P D and p - N A D activities. The signif icant difference in the levels of mono-oxygenase activi- ties between the two species at t ime 0 of pre- in- cubat ion , suppor t s the fact that endogenous G-6- P D H is much higher in the $9 fract ion from rat.

The s tabi l i ty da ta show an enhanced decrease in the rate of A P D and p - N A D activit ies in the rat wi thout exogenous G - 6 - P D H . This is p r o b a b l y due to the paral le l loss of G - 6 - P D H . These results are in agreement with the l i terature; in fact, M a m a e v et al. (1977), using similar condi t ions of incubat ion, demons t ra t ed a loss of 30% of G-6- P D H activi ty in 25 rain.

By evaluat ing the s tabi l i ty da ta in the presence of exogenous G - 6 - P D H , p - N A D activi ty appears to be more s table than A P D in the mouse, as repor ted by Paol ini et al. (1983). In contrast , in the rat, A P D activi ty was more s table than p - N A D .

296

The ED activity was less stable than APD and p-NAD activities both in the mouse and in the rat. As the isoenzyme composition of the cytochrome P-450 complex is partly reflected in the various mono-oxygenase activities measured, the observed differences indicate certain species differences in isoenzyme composition following pretreatment of the studied species with the various inducers. Our results suggest that the presence of exogenous G-6- PDH in LMA incubation mixtures, increases the N A D P H / N A D P ÷ ratio, and consequently enhances the specific isoenzyme activities underly- ing the various phase I enzyme activities studied, and reduces the competitive inhibition exerted by NADP ÷ on NADPH cytochrome c reductase (Gil- lette, 1971). However, at the same time, enhance- ment of NADPH-dependent lipid peroxidation may decrease the mono-oxygenase stability.

The mitotic gene-conversion and mitotic cross- ing-over by DMN and AR2MNFN on the D 7

strain of Saccharomyces cerevisiae increased when $9 fraction (mouse or rat) and exogenous G-6-PDH were added to the incubation mixtures for LMA. Thus, for detecting the potential mutagen re- sponse, it is useful to add, systematically, in in- cubation mixtures, exogenous G-6-PDH combined with $9 fraction for bioactivation of a unknown xenobiotic. Clearly, exogenous G-6-PDH increases the hepatic cytochrome P-450 mono-oxygenase system of the post-mitochondrial supernatant ($9 fraction) of mouse and rat, during incubation for mutagenic assay.

In conclusion, this work contributes to an un- derstanding of the biochemical events underlying the mono-oxygenase activity and particularly, mono-oxygenase stability; moreover it adds in- formation in order to clarify the complex biochem- ical phenomena which occur during incubation for drug biotransformation in 'in vitro' mutagenicity tests.

Acknowledgements

We are grateful to Professor Carlo Bauer of the Institute of Biochemistry, Biophysics and Genet- ics, University of Pisa (Italy), who read a pre- liminary draft of this paper and provided helpful suggestions.

This work was supported by C.N.R. (National

Research Council of Italy) grants, Applied Project 'Preventive and Rehabilitative Medicine' and ' Oncology'.

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