Developmental changes in hepatic activation of dietary mutagens by mice

Chem.-BioL Interactions, 71 (1989) 367--379 Elsevier Scientific Publishers Ireland Ltd.

367

D E V E L O P M E N T A L C H A N G E S IN H E P A T I C A C T I V A T I O N O F D I E T A R Y M U T A G E N S BY M I C E

W.E. BRENNAN-CRADDOCK~b, S. NEALE', A.K. MALLETT b and I.R. R0WLAND b

°Department of Biochemistry, University College, Londo~ WCIE 6BT and bDepartment of Microbiology, The British Industrial Biological Research Associatio~ Carshaltwf~ Surrey, SM5 ~DS fU.K.) (Received October 10th, 1988) {Revision received February 2nd, 1989) {Accepted February 21st, 1989)

SUMMARY

Metabolic activation of the food mutagens 2-amino-3,4-dimethylimidazo[4,5f]quinoline (MeIQ), 3-amino-l-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and ariatoxin B 1 by female BALB/c mice of different ages ( 2 - 2 4 weeks) was investigated in vivo and in vitro using Salmonella typhimurium TA98 as the indicator organism. The in vivo activation of the three mutagens was investigated in 4- and 24-week-old mice using an intrasanguineous host-mediated assay. All three compounds showed reduced levels of activation with the older hosts. Hepatic $9 fractions from female mice of varying ages between 2 and 24 weeks were used in the in vitro mutagenicity assay. To achieve optimal activation to bacterial mutagens, 5% $9 was required for aflatoxin B 1 and Trp-P-2 and 10% $9 for MeIQ; age of donor generally had little effect on the profile of these protein activation curves. Under these optimal conditions MeIQ and Trp-P-2 both exhibited, as before, age-dependent decreases in activation over a wide range of mutagen concentrations, however the in vitro activation of aflatoxin showed no consistent change with age. Spectrophotometric measurements of $9 cytochrome P-450 content showed a decrease in concentration with increasing age, but this was not sufficient to account for changes observed in hepatic mutagen activation. However, changes in the activities of certain cytochrome P-450 isoenzymes and cytosolic GSH-transferases, which in turn result in changes in the activation and detoxification capacity of the liver, would appear to explain age-dependent changes in the activity of mutagens in vivo.

Key words: Age -- In vivo/in vitro mutagenicity -- Dietary mutagens -Hepatic metabolism

0009-2797/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

368 INTRODUCTION

Cytochrome P-450-dependent mixed-function oxidase (MFO) pathways, localised in tissue microsomal fractions, play an important role in the metabolism of many endogenous substances or xenobiotic compounds which gain access to the body [1]. The activity of the hepatic enzyme system exhibits marked age-related changes [2] which may alter the disposition of toxic or pharmacologically-active substrates in vivo. The haemoprotein and flavoprotein components of MFO are initially absent, or very low, in the foetus and neonate, but generally increase together with their respective activities during the immediate perinatal period [3]. However, the various isoenzymes which comprise the cytochrome P-450 exhibit different developmental patterns [4,5]. For example, the hydroxylation of 4-methylcoumarin in rats reaches a peak 3 - 5 days postpartum and then declines to adult levels [6], while the metabolism of other substrates are maximal 2 weeks [7], 1 month [8] or 6 months [9,10] after birth. Certain MFO activities stabilise in adult animals, and give approximately constant values at 1 or 2 years of age [11], while the expression of other functions decreases in adult or geriatric animals [2,9]. A coherent pattern for the development of cytochrome P-450-associated pathways is clearly lacking and may reflect the influence of diet [12,13], hormones [9,14], species and strain differences on enzyme synthesis and expression. A large number of carcinogens and mutagens are dependent on mammalian xenobiotic-metabolising enzymes for their genotoxic activity, which may exhibit age-related changes, as demonstrated by Raineri et al. [15]. For example, the activation of 3-methylcholanthrene or benzo[a]pyrene by Aroclor-induced rat hepatic $9, and N-nitrosopyrrolidine by male hamster preparations, decreases with increasing donor age [15]. This may in turn lead to differential susceptibility of animals at various stages of their development to the mutagenic or carcinogenic effects of such compounds. Electrophilic intermediates of xenobiotics produced by the MFO system may be metabolized by conjugation to glutathione (GSH). No difference in the hepatic GSH content is apparent between neonatal and adult mice or rats [16,17], but marked age-related changes can be observed in the activities of hepatic GSH-transferases, the group of enzymes which catalyse the conjugation with GSH [18]. Generally, the hepatic GSH-transferase activities in mice and rats increase from a basal neonatal level and reach adult levels by 5 weeks of age, but the rate of increase varies between the different transferases, as reflected by the substrate used [4,19]. GSH and GSH-transferases are thought to provide an important detoxification pathway for aflatoxin B1 [20] and N-hydroxy-Trp-P-2 (the proposed ultimate mutagen of Trp-P-2) is also able to conjugate with GSH [21]. In this study we report the in vivo activation of dietary mutagens and carcinogens by 4- and 24-week-old mice using a host-mediated assay and compare these results with those for in vitro activation of the compounds by

369

hepatic fractions from mice aged between 2 and 24 weeks. Results for the heterocyclic amine mutagens 2-amino-3,4-dimetbylimidazo[4,5-]]quinoline (MeIQ) and 3-amino-l-methyl-5H-pyrido-[4,3-b]indole (Trp-P-2), which are found in heated proteinaceous food, and the fungal metabolite aflatoxin B1 are presented. All three compounds are dependent on activation by mammalian hepatic enzymes for their genotoxic activity [21,22]. Complementary results on age-related changes in cytochrome P-450 content and function and GSHtransferase activities are also described. MATERIALS AND METHODS

Animals Pregnant female inbred BALB/c mice were purchased approximately 3 weeks after mating from Harlan Olac Ltd. (Bicester) and fed a stock breeding diet (Rat and Mouse Diet No. 3, Special Diet Services, WithamL At birth, the mothers were transferred onto a purified, fibre-free diet [23] and the offspring were weaned onto this at 3 weeks of age. Diet and tap water were available ad lib. until the animals were killed.

Host-mediated assay A modification of the intrasanguineous host-mediated assay by Arni et al. [24] was used. An aliquot of 0.1 ml stationary phase S. typhimurium TA98 suspension in saline ( 4 - 6 x 101° cells/ml) was injected intravenously via the tail vein immediately prior to a p.o. dose of either aflatoxin B1 (10 mg/kg), MeIQ (2.5 mg/kg) or Trp-P-2 (10 mg/kg) given as 0.01 ml mutagen/g body weight. The mutagens were dissolved to the required concentrations in dimetbylsulfoxide (DMSO). Control animals received 0.01 ml DMSO/g body weight. One hour after bacterial injection the mice were killed by cervical dislocation and their livers removed and homogenized aseptically in 10 ml 50 mM Tris/150 mM KC1 using an Ultra-Turrax homogenizer. After initial centrifugation for 10 rain at 100 × g the resulting supernatant was centrifuged at 1700 x g for 30 rain and the sedimented bacterial cells were then resuspended in 1 ml Tris-KCl. To determine the number of revertants, 2.5 ml top agar (50°C) containing histidine (0.1 ~mol), biotin (0.1 ~mol) and ampicillin (0.14 retool) were added to 0.1 ml of bacterial suspension in triplicate and the mixture was poured onto 20 ml Vogel Bonner agar plates. The number of bacterial survivors was determined by plating 0.1-ml aliquots of a suitable dilution (10-e) onto nutrient agar plates. All plates were incubated at 37 °C for 48 h.

Preparation of $9 fractions Mice were killed by cervical dislocation at 2, 4, 6, 8, 12 and 24 weeks. The livers from at least 3 animals of similar age were pooled and homogenized aseptically in Tris--KC1 (pH 7.4) (4 vol./g liver} using five reciprocal strokes of a Potter-Eveljhem tissue homogeniser. Hepatic post-mitochondrial ($9) fractions were prepared by differential centrifugation (9000 × g for 20 rain)

370 and small aliquots (2 ml) of each were immediately snap-frozen in sterile tubes by immersion in liquid nitrogen and stored at - 8 0 °C until required. For determination of MFO and GSH-transferase activities of 4 and 24week-old mice, samples of $9 from these age groups were centrifuged at 104 000 × g for 1 h. The resulting supernatant (cytosol) was stored at - 8 0 ° C until later use. The microsomal pellet was resuspended to the original liver weight with Tris-- KC1. Protein concentrations were determined by the method of Lowry et al. [25].

In vitro mutagenicity assay In all experiments, the Salmonella mutagenicity assay was performed at 37 °C with a preincubation step of 30 min, based on the method of Maron and Ames [26]. Each incubation tube contained (in a total volume of 0.8 ml): 2 ~mol ~-nicotinamide adenine dinucleotide phosphate, 1 ~mol D-glucose 6-phosphate, 16.5 ~mol KCI, 4 ~mol MgC12, 50 ~mol NaH2PO 4 buffer (pH 7.4), $9 fraction and 0.1 ml stationary phase S. typhimurium TA98 (approximately 1 x l0 s cells). As glucose-6-phosphate dehydrogenase may be limiting in the younger animals, 1.08 units was added to each tube. Tris--KCl was used to adjust the incubation volume as necessary. After the incubation period, molten top agar (50°C) containing histidine (0.1 ~mol) and biotin (0.1 ~mol) was added to each tube and the mixture overlaid onto 20 ml Vogel Bonner agar plates. The plates were incubated for 48 h at 37 °C and the number of mutants was scored using an 'Artek' colony counter (Artek Systems Corp., New York, U.S.A.). All assays were done in triplicate. In preliminary studies, different amounts of $9 fraction in the incubation mixture were compared for their ability to activate MeIQ, Trp-P-2 and aliatoxin B 1. The concentration of $9 fraction that induced the maximum number of revertants for each mutagen was used in subsequent experiments with a range of mutagen concentrations. Determination of cytochrome P-450 The cytochrome P-450 content of the $9 and microsomal fractions was determined from the carbon monoxide-saturated dithionite difference spectrum as described by 0 m u r a and Sato [27]. Determination of cytochrome c reductase The determination of this microsomal flavoprotein enzyme was based on the principle that when oxidized cytochrome c is converted to reduced cytochrome c it has, unlike the former, a characteristic absorption maximum of 550 nm. (Method as described by Lake [28]). Determination of MFO enzyme activities Activities of 7-ethoxycoumarin- and 7-ethoxyresorufin-O-deethylase in hepatic microsomal fractions were measured by fluorescence intensity as described by Lake [28]. N-Demethylation of benzphetamine was determined by the method of Lu et al. [29] based on the measurement of formaldehyde

371 production. The formaldehyde is trapped in the incubation medium by the presence of semicarbazide and subsequently estimated colorimetrically by means of the Hantysch reaction [30].

Detezmination of total GSH, GSSG and GSH-tzansferase Total giutathione (GSH) and oxidized giutathione (GSSG) from the whole homogenate were measured by the method of Adams et al. [31]. GSH-transferase activities in the hepatic cytosol were estimated by the method of Habig et al. [32] using 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4nitrobenzene (DCNB) and 1,2-epoxy-3~/~nitrophenoxy)propane (ENPP) as substrates. Analysis of results Statistical analysis was carried out by analysis of variance using the Minitabs Statistical Package (Minitals Inc., PA). Significant differences between mean values were assessed using the least significant difference criterion [33]. RESULTS

Host-mediated assay The activation of Trp-P-2 (10 mg/kg), MeIQ (2.5 mg/kg) and aflatoxin B 1 (10 mg/kg) to bacterial mutagens by female BALB/c mice in the host-mediated assay was greater by 121%, 49°/0 and 28% respectively in 4-week-old mice compared to mice 24 weeks of age (Table I). The reversion to histidine auxotrophy by S. typhimurium TA98 in the control mice given DMSO only, remained at levels equivalent to the spontaneous mutation frequency in the original bacterial suspension (Table I). The recovery of bacteria from the liver was similar in all treatment groups.

TABLE I EFFECT OF AGE ON ACTIVITY OF FOOD MUTAGENS IN MURINE HOST-MEDIATED ASSAY Female mice of 4 and 24 weeks of age were exposed to mutagens and S. typhimurium TA98 in the host-mediated assay, as described in Materials and Methods. Values given as mean ± S.D. Number in parentheses. Age

Body wt.

His°/Plate

(weeks)

(g)

Controls DMSO only

AFB 1 10 mg/kg

MeIQ 2.5 mg/kg

Trp-P-2 10 mg/kg

8 ± 4 (4) 8 ± 3

230 ± 47 (8) 179 ±54"

2950 ± 521 (7) 1974 ± 869~

1753 ± 238 (6) 793 ± 253"

(4)

(8)

(8)

(8)

4

9.4 ± 1.5

24

19.6 ± 1.7

•Significantly different from values in the 4-week age group at P < 0.001.

372

In vitro mutagenicity assay Mouse liver weights increased rapidly over the first six weeks of life (Table II) concomitant with the changes in body weight (6 g rising to 14 g) over that period. Subsequently the rate of increase in liver and body weight declined. The amount of $9 protein present in the hepatic post-mitochondrial supernatant was lowest for preparations from the 2-week-old mice and increased with increasing donor age up until around week 8 postpartum (Table II). However, the concentration of cytochrome P-450/g of liver remained approximately constant in all mice throughout the study, although the specific content of haemoprotein/mg $9 protein exhibited an age-related decrease between weeks 6 and 8 (Table II). The concentration of $9 in the incubation mixture required for optimum activation of MeIQ to a bacterial mutagen was 10O/o of the final volume (equivalent to 2 - 3 mg $9 protein/ml incubation mix) irrespective of the age of the mice (Fig. 1). For Trp-P-2 and aflatoxin B1 the optimal concentration of $9 was 50/0 (equivalent to 1--1.5 mg $9 protein/ml incubation mix). Again, the optimal concentration of $9 was similar over the age range of animals used (Fig. 1). These optimal $9 concentrations were used throughout subsequent experiments. No differences in spontaneous mutation frequency of S. typhimurium TA98 were seen in the presence of varying concentrations of $9 fractions or $9 fractions from mice of different ages (data not shown). The hepatic activation at any given concentration of MeIQ was maximal when $9 from 2- or 4-week-old mice was used and in general the activity of the mutagen declined as the age of the animals increased (Fig. 2). Similar results T A B L E II

PROTEIN AND CYTOCHROME DIFFERENT AGES

P-450 C O N T E N T

OF $9 F R A C T I O N S F R O M

M I C E OF

Hepatic $9 p r o t e i n a n d c y t o c h r o m e P-450 c o n t e n t s w e r e d e t e r m i n e d in female BALB/c mice of d i f f e r e n t ages, as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . Values g i v e n as m e a n ± S.D. N u m b e r in p a r e n t h e s e s . E a c h replicate s a m p l e c o n t a i n e d livers pooled from 4 mice (7 in t h e case of t h e 2w e e k ~ l d pre-weaners). Age w e e k s (n)

2 4 6 8 12 24

(4) (4) (4) (4) (4) (6)

L i v e r wt. (g)

0.24 0.50 0.76 0.86 0.85 0.88

± ± ± ± ± ±

0.04 0.08 0.07 0.19 0.03 0.12

$9 P r o t e i n

C y t o c h r o m e P-450

m g / g liver

n m o l / m g $9 protein

nmol/g liver

77 87 90 118 99 116

16.3 16.6 17.1 12.6 13.8 17.0

0.21 0.19 0.19 0.11 0.14 0.14

_+ _+ _+ _+ _ ±

5 8 7 14 ~ 4b 10 b

_ _+ _+ ± +_ ±

2.0 1.6 1.7 3.5 2.4 4.8

•Significantly d i f f e r e n t v a l u e s from t h e p r e v i o u s a g e g r o u p at P ~ 0.001 level. bSignificantly different v a l u e s at t h e P ~ 0.05 level.

_+ _+ ± ± ± ±

0.03 0.01 0.01 0.03 ~ 0.02 0.03

373

MelQ (0.047 nmol ) 1200

Trp-P-2 ( 0.12 nmol) o

00.

o/"Oo

o

.,,..,,.

800

q/o A

Q

Aflatoxin B1 ( 0.64 nmol) 4.50

o~O

d

00-

"C

l

~D

E +

400

200

°

I

n

15o

°~

"-r-

0

I

I

0

10

I

20

0

I

I

0

10

I

20

0

I

!

0

I0

I

20

% $9 in assay Fig. 1. Determination of optimal $9 concentration for mutagen activation in vitro. Different concentrations of hepatic $9 expressed as % of incubation volume (0.8 ml) from mice aged 4 weeks (O), 8 weeks (1"1)or 24 weeks CA) were incubated with the appropriate mutagen and the indicator organism S. typhimurium TA98 for 30 rain at 37°C before being poured, in top agar, onto VogelBonner plates and incubated for 48 h at 37°C. Results are given as means for three pooled $9 preparations. Each incubation was performed in triplicate.

were obtained for Trp-P-2 although, in this case, the mutagenicity in the presence of $9 from 2-, 4- and 6-week~ld mice remained unchanged followed by a marked fall in activating capacity between 6 and 12 weeks (Fig. 2). In contrast to the results for the two cooked-food mutagens, the activation of aflatoxin B 1 did not decrease with age of animal and tended towards a slight upward trend with age (Fig. 2). The greater activation of MeIQ by hepatic $9 from young mice (4-weekold) was apparent over a wide range of mutagen concentrations (0.02-0.2 nmol), with an approximate doubling in the numbers of revertants generated when compared with incubations containing $9 from 24-week-old mice (Fig. 3). Trp-P-2 also was more active when used with liver preparations from 4week-old mice with, in this instance, a 2.5-fold excess in reversion to histidine prototrophy relative to the activating capacity of the older mice (Fig. 3). In contrast, aflatoxin B 1, at all concentrations studied, was equally well metabolised to a mutagen by mouse hepatic fractions irrespective of donor age (Fig. 3). MFOs No difference was observed in cytochrome P-450 content (nmol/mg prot.)

o L

0

1200

24OO

3600

800-

1600

'

0

'0

'

;~ 1'6

2o

4wk

0

700

1400

300

600'

0

900-

1200-

2100

O.07nmol

74

' o.'20 0.25

nmol MeIQ/assay

'

MelQ

Age (weeksl

'8

t

0.05 0.10 0.15

i

e ~

)}~}~ MelQ

i

0

i

i~

i;

0.1 '

Trp-P-2 ~

~

O.i 5

Age (weeks)

i

8

nmol Trp-P -2/assay

'o

O. 5

i

4

t.,~ t~ I

\

Trp-P-2

4wk

0.2 '

i

2o ~4

~ O.12nmol

800

1200

o f-0

50O

750-

I~-

r 0.5

i'2

~

i

BI

] 1.5

i'6 20 2~ Age (weeks)

i

8 Aflatoxin

~

nfaol aflatoxin BI/assay

1

o

~.i.{__~/j{

Aflatoxin B1

O3,nmo,

Fig. 3. Activation of mutagens by hepatic $9 from 4- and 24-week-old mice. For details see legend to Fig. 2.

Fig. 2. Activation of mutagens by $9 fractions from mice of different ages. Mutagen, cofactors, S. typhimurium TA98, and $9 fractions were incubated for 30 min at 37°C before being poured, in top agar, onto Vogel-Bonner plates and incubated for 48 h at 37°C. The concentration of $9 fraction in the incubation mixture was 5% (v/v) for Trp-P-2 and aflatoxin B1 and 10% (v/v) for MeIQ. Results are expressed as means (bars indicate S.D.) for four $9 preparations. Each incubation was performed in triplicate.

i

+

i

+

"E

o

2400-

3200-

e~

79.42 ± 5.6 71.76 ± 9.1

1704.8 ± 189 1805.7 ± 125

(D)

(C)

13.41 ± 2.2 18.65 ± 3.6b

(B)

(A)

0,029 ± 0.12 0.701 ± 0.07

Cytochrome c reductase

Cytoelirome P-450

10420 ± 11.5 121.74 ± 15.5

(E) 2234.5 ± 278 3228.0 ± 390"

(F)

Ethoxyeoumarin-O-deethylase

28.53 ± 3.3 27.21 ± 6.1

(G) 614.36 ± 99.1 708.07 ± 99.3

(H)

Ethoxyresorufin-O-deethylase

0.255 ± 0.09 0.256 ± 0.09

(I)

5.46 ± 1.8 5.72 ± 0.7

(J)

Benzphetemine demethylatinn

36.50 ± 5.6 32.42 ± 9.1

(A)

7.66 ± 1.11 7.38 ± 1.22

(B)

Total glutathione (D)

0.043 ± O.Ol 9.02 ± 2.0 0,030 ± 0.01 6.88 ± 0.8

(C)

GSSG

•Significantly different from values in the 4-week age group at P = 0.001. bSignificantly different at P < 0.05. eSignificantly different at P < 0.01.

4 (6) 24 (6)

(weeks)

Age

0.050 ± 0.2 0.060 ± 0.01

(E)

DCNB

5.26 ± 1.92 6.72 ± 1.81

(F)

1.566 ± 0.26 2.184 ± 0.85b

(G)

CDNB

164.8 ± 26.2 240.67 ± 34.5"

(H)

0,227 ± 0.05 0.163 ± 0.05¢

(I)

ENPP

23.88 ± 5.3 17.89 ± 4.9

(J)

Measurements of total GSH content, GSSG and the activities of GSH-transferases towards the substrates DCNB, CDNB and ENPP were determined in hepatic fractions of female BALB/¢ mice of 4 and 24 weeks of age, as described in Materials and Methods. (A) nmol/mg protein; (B) mM; (C) nmol/mg protein; (D) raM; (E) pmol/min/mg protein; (F) pmo[/min/g liver; (G) ~mol/min/mg protein; (H) ~4mol/min/gliver; (1) ~mol/min/mg protein; (J) ~Lmol/min/gliver. Values are given as mean ± S.D. Number in parentheses. Each replicate sample contained livers pooled from 6 mice.

DETERMINATION OF HEPATIC LEVELS OF GSH AND GSH-TRANSFERASES IN MICE AGED 4 AND 24 WEEKS

TABLE IV

•Significantly different from the values for the 4-week age group at P = 0.001. bSignificantly different at P ~ 0.01 level.

4 (6) 24 (6)

Age (weeks)

(A) nmoljmg protein; (B) nmol/g liver; (C) nmol/minlmg protein; (D) nmol~ninlg liver; (E) nmol/h~ng protein; (F) nmol/h/g liver; (G) nmol/h/mg protein; (H) nmol/h/g liver; (I) pmol/h/mg protein; (J) p~noll hlg liver. Values are given as mean ± S.D. Number in parentheses. Each replicate sample contained livers pooled from 6 mice.

CYTOCHROME P-450, CYTOCHROME c AND MFO ACTIVITIES OF MOUSE MICROSOMAL TISSUE AGED 4 AND 24 WEEKS

TABLE HI

376 or cytochrome c reductase activity (nmol/min per mg prot.) in the hepatic microsomal fractions from mice aged 4 and 24 weeks, but the older mice showed a 40% increase in total cytochrome P-450 content when the results were expressed as per g liver. The three MFO activities showed little difference when expressed as per mg protein (Table III). However, ethoxycoumarin-O-deethylase activity/g liver was significantly higher (1.5fold) for the 24-week-old mice with a similar small, although not significant, increase in the expression of ethoxyresorufin-O-deethylase activity.

GSH and GSH-transferases No marked age-related difference was found in the total GSH content present in the whole homogenate of liver from mice aged 4 and 24 weeks. GSSG content, however, was reduced in the older hosts (Table IV). Similar GSHtransferase activities were recorded for the two age groups when DCNB and E N P P were used as substrates; however, using CDNB, GSH-transferase activity was 1.5-fold greater in the older hosts (Table IV). DISCUSSION

Marked age-related differences were detected in the in vivo activity of three potent dietary mutagens. MeIQ, Trp-P-2 and aflatoxin B 1 (Table I). The results suggest that young animals may be more susceptible to the genotoxic effects of these compounds than older animals. It is noteworthy that long-term, rodent bioassays of other food-borne carcinogens which require metabolic activation, notably N-nitroso derivatives of diethylamine and dimethylamine [34,35] have also revealed age-dependent changes in tumour incidence; with rats exposed from 3 weeks of age exhibiting a 20-fold higher incidence of liver tumours than those in which exposure began at 20 weeks [35]. The in vitro mutagenicity studies, using optimal concentrations of hepatic fractions from mice of different ages, strongly suggest that the developmental changes in in vivo mutagenicity of MeIQ and Trp-P-2 are attributable to age-dependent changes in the metabolism of the mutagens. Similar decreases in in vitro mutagenicity with increasing age have been reported for 3-methylcholanthrene, benzo[a]pyrene and N-nitrosopyrrolidine [15]. However, contrary to the in vivo data the in vitro mutagenicity of ariatoxin B1 was not affected by age. This is in accord with the results of Jayaraj et al. [36], who reported that aflatoxin activation in rats remained unaltered up until 12 months of age, although Robertson and Birnbaum [37] recorded a decline in activation for rats by the age of 2.5 months. These conflicting results may be attributable to rat strain and species differences. The decrease in mutagenicity with age may be due to a decrease in the enzymes which initially activate the chemical to its electrophilic reactive species, which are believed, for MeIQ, Trp-P-2 and afiatoxin B 1, to be the cytochrome P-450-dependent MFOs [2,38]. However, the change in the hepatic cytochrome P-450 content with age (Table III) did not follow closely the changes in Trp-P-2 and MeIQ activating capacity. Since the microsomal

377 proteins (in which the cytochrome P-450 resides) comprise only 200/0 of the total $9 protein it is possible that developmental changes in the soluble protein may distort measurements of cytochrome P-450 when expressed in terms of $9 protein. However, it should be noted that cytochrome P-450 levels measured in hepatic microsomes (Table III) do not closely follow the changes in activating capacity of Trp-P-2 and MeIQ either. A possible explanation for this is that the content of total hepatic cytochrome P-450 may not accurately reflect the levels of specific haemoprotein isoenzymes involved in the activation of these mutagens, the expression of which are developmentally regulated [14]. Heterocyclic amines such as MeIQ and Trp-P-2 are thought to be activated specifically by cytochrome P-448-dependent N-hydroxylation [21] and may also play a role in the activation of aflatoxin B1 [22]. The O-deethylation of ethoxyresorufin (which is used to monitor cytochrome P-448 activity [39]) was found not to change significantly with age. However, it has been found that this enzyme activity preferentially reflects the activity of a low spin form of cytochrome P-448, whereas N-hydroxylation of cooked food mutagens involves a high spin species [21]. It is possible, therefore, that microsomal levels of this high spin form of cytochrome P-448 decrease with age and are responsible for the observed changes in the mutagenicity of MeIQ and TrpP-2. Alterations in the detoxification (Phase II metabolism) of these compounds may also play a role in age-related changes in genotoxicity since an increase in the activity of the GSH transferases that use CDNB as a substrate was also detected as the age of the animals increased. CDNB acts as a suitable substrate for a wide range of GSH-transferases, whereas DCNB and ENPP, in which no age-dependent change was found, are more enzyme-specific [40]. Little is known about the enzymes involved in the conjugation of MeIQ and Trp-P-2 metabolites to GSH, although N-hydroxy Trp-P-2 (the proposed ultimate carcinogen of Trp-P-2) is able to form three GSH conjugates [41]. Conjugation to GSH is a major detoxification pathway of aflatoxin B1 [20] and, since hepatic $9 primarily measures the activation of compounds, changes in this phase II metabolic pathway may explain age-related differences in aflatoxin B1 mutagenicity observed in vivo but not in vitro. It was considered unlikely that the differential effects of age on in vivo mutagenicity found in our studies were a consequence of differences in the rate of intestinal absorption of foreign compounds from the gut of mice in the two age groups. The uptake of MeIQ from the small intestine of adult mice is extremely rapid, with peak levels being measured in the blood and liver only 10 rain after injection of MeIQ into ligated gut sections (Howes and Rowland, unpublished observation, 1988). Overall, our results indicate that the in vivo mutagenicity of certain dietary genotoxins is markedly influenced by the age of the animal and that in the case of the cooked-food mutagens MeIQ and Trp-P-2, these changes may be due primarily to developmental changes in the enzymes involved in the activation of these compounds, while for aflatoxin B1 the conjugation of the reactive species to glutathione would appear responsible.

378 ACKNOWLEDGEMENTS

The authors would like to thank Dr B.G. Lake for his assistance in determining GSH and GSSG content and GSH-transferase activities. W.E. Brennan-Craddock would like to thank SERC for their CASE award. A.K. Mallett and I.R. Rowland were funded by the U.K. Ministry of Agriculture, Fisheries and Food and S. Neale by the C.E.C. Contract no. ENV-536-UK(H). REFERENCES 1 2 3

4 5

6 7

8 9

10 11 12 13 14 15

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17 18

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