Chent-Biol. Interactions, 15 (1976) 107--116 107 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
INFLUENCE OF LEAD NITRATE Ol~l DIMETHYLNITROSAMINE INTOXICATION
P. PANI, A. COLUMBANO, S. DESSI, M. PORCU and L. CONGIU Istituto di Patologia Generale, Universitb di Cagliari (Italy) (Received March 20th, 1976) (Accepted May 27th, 1976)
SUMMARY
The effect of lead nitrate, an inhibitor of the hepatic drug-metabolizing enzyme system upon the acute hepatotoxicity of dimethylnitrosamine (DMN) was studied. Lead pretreatment significantly prevented polysomal disaggregation induced by the nitrosamine. Cell necrosis, evaluated morphologically and by the release of serum glutamic-pyruvic transaminase (GPT), was also diminished. The metabolism of DMN in rats pretreated with lead nitrate was investigated by following its clearance from blood and by determining, in vitro the demethylation of the nitrosamine. Lead increased, although not significantly, the clearance of DMN from blood, but it lowered the activity of DMNdemethylase 24 h after its administration. Finally, lead lowered the lethal effects of DMN. The mechanism by which lead influenced DMN toxicity is discussed.
INTRODUCTION
The increasing interest in the field of biotransformation of chemical compounds occurring in the hepatic oxidative chain of the smooth endoplasmic reticulum has le~t several authors to study the alterations which, by modifying the system, may affect the toxicity of those compounds which are metabolized by it [ 1]. The drug-metabolizing enzyme system may he affected by several factors, including environmental pollutants [2]. Among these, lead is spread through the world in concentrations found to be continuously increasing [3]. ReAbbreviations: DMN, dimethylnitrosamine; GPT, glutamic-pyruvic transaminase.
108 cently Alvares et al. [4] and Scoppa et al. [5] have found that a single dose of lead decreases the activity of the drug-metabolizing enzyme system 24 h after its administration to the rat. It has been shown that lead exerts a protective effect against CC1,-induced hepatotoxicity [6]. DMN, as in the case of CC14 [7,8], is also metabolised by oxidative microsomal enzymes into derivatives which induce hepatic damage as well as being carcinogenic [9]. A relationship between the pathophysiological state of the drug-metabolizing enzyme system and DMN-toxicity has not yet been completely cleared up [10,11]. Protection against DMN-acute toxicity has been attempted by inhibiting the drug-metabolizing enzyme system [ 12--14 ]. Our experiments have been performed to study the action of lead on some pathological alterations induced by DMN, that is polysomal disaggregation and cell necrosis. We have followed the metabolism of DMN by measuring the disappearance of DMN from blood and, indirectly, by measuring DMNdemethylase. Some preliminary results were previously shown [ 15 ]. MATERIALS AND METHODS
Animals. Male Wistar rats (200--250 g) were used in these experiments; they were fed a semisynthetic diet (Ditta Piccioni, Brescia, Italy), free of antioxidants. Treatments. Lead nitrate, dissolved in 0.9% NaC1, was injected intravenously, under light ether anaesthesia (100 pmoles/kg b.w.) 24 h before DMN-intoxication. Control rats received saline only. Dimethylnitrosamine (Merck, Darmstadt, Germany), dissolved in 0.9% NaC1, was injected intraperitoneally at different doses (see RESULTS). Control rats received saline only. Analytical procedures. For the determination of the polysomal profiles, rat livers were homogenized, 20% (w/v) in a medium (TKM buffer) containing 0.15 M sucrose, 0.05 M Tris--HC1 buffer pH 7.8, 0.025 M KC1, and 0.005 M MgCI~, in a Potter--Elvehjem homogenizer fitted with a Teflon pestle. The postmitochondrial supernatant was prepared by centrifuging the homogenate at 15 000 g for 10 min; it was then treated with 1% sodium deoxycholate. 0.4 ml of the deoxycholate post-mitochondrial supernatant was layered over a 5.5 ml linear gradient of 15--50% sucrose, obtained by using a Gradient Former, Model 570, ISCO (Lincoln, Nebraska, USA). The gradients were then centrifuged at 130 000 g for 60 min with the SW 50 rotor of a Beckman-Spinco, Model L 50, preparative ultracentrifuge. Serum activity of GPT was determined according to a standard combination method provided by Boehringer (Mannheim, Germany). The determination of the activity of the DMN-demethylase was performed according to the method of Venkat~san et al. [16]. 50% homogenates Of the liver were prepared in ice-cold 0.25 M sucrose; nuclei and mitochondria were removed by centrifugation at 9000 g for 20 min. The microsomes were sedimented at 105 000 g for 60 rain with the 50 rotor of a Beckman-Spinco, Model L 50, preparative ultracentrifuge. The microsomalpellet was finely
109 suspended by using a Polytron homogenizer (Kinematica, G m b H , Luzern, Switzerland) at position 3 for 5--10 sec in ice-cold 0.1 M K H 2 P O 4 buffer p H 7.4. Incubation was carried out in an open erlenmeyer flask at 37°C in a Dubnoff metabolic shaker. The reaction mixture contained: MgCI2 (20 /~moles), niacin (40 #moles), semicarbazide (75/~moles), N A D P (4 #moles), NADPH-generating system consisting of glucose-6-phosphate (40 /~moles) and glucose-6-phosphate dehydrogenase (4 Kornberg units), p H 7.4 phosphate buffer (3 mmoles), D M N (40/~moles), microsomes from 1.8 g of liver and 1.15% KCI to bring the volume to 10.0 ml. The demethylation was determined from an aliquot of the reaction mixture after 30 rain incubation. Formaldehyde was determined by the method of Nash [17], modified by Cochin and Axelrod [18]. Proteins were determined by the method of Lowry et al. [19]. For the D M N plasma concentration, 3 ml of blood was withdrawn from the abdominal aorta of rats under light ether anesthesia. D M N was determined by the method of Heath [20] with slight modifications. The blood was poured into 50 ml centrifuge tubes containing 20.0 ml of 2.5% sulphosalicylic acid. Samples were centrifuged and filtered into tubes containing 0.3 ml 12.5 N N a O H . To a 15.0 ml aliquot 2--3 g of N a O H , in pellets,was added. 9.0 ml were distilled,and the distillatewas diluted to 10.0 ml with distilled water. 1.0 ml of 2.5% sulphosalicylicacid was added. Polarographic determinations of D M N were performed using a P O 4 Polariter (Radiometer, Copenhagen, Denmark). Histological technique. After removing the liver,small portions were immediately fixed in Bouin's solution, embedded in paraffin, and stained with hematoxylin~osin. RESULTS Table I shows the mortality induced by DMN in rats pretreatecl with lead nitrate or with DMN alone. Lead nitrate appeared to affect lethality in two ways: b y decreasing the absolute number of dead animals and b y delaying the time of death. It was previously seen that an inducer of the drug-metabo-
TABLE I PROTECTION BY L E A D NITRATE AGAINST LETHALITY INDUCED BY D M N Lead nitratewas given at a dose of 100 pxnoles/kgb.w. by intravenousinjection24 h before D M N administration.D M N was givenat a dose of 40 mg/kg b.w. Treatment
Number of
Survivors
animals
Lethality (%)
Deaths at 3 days Total deaths (%)
DMN Pb + D M N
27 24
8 13
70 46
63 20
II0 lizing enzyme system, phenobarbital, hastened the onset of the death in rats given DMN, while an inhibitor, SKF 525-A, did not influence the mortality caused by the nitrosamine [21]. The effect of lead pretreatment on polysomal disaggregation is shown in Fig. 1 and Table II. Lead nitrate clearly protects against the strong polysomal disaggregation caused by DMN. The rats treated with lead alone did not show any alteration, as was previously shown [6]. The level of the serum GPT is greatly reduced in the lead pretreated group compared with the high values for rats treated with DMN alone (Table II). In the livers of rats treated with DMN alone a severe zonal necrosis appears, which is mainly centrolobular, and associated with hemorrhage and sinusoidal congestion. Scattered cells, bordering the necrotic areas, showed ballooning (Fig. 2a). On the other hand, livers from rats pretreated with lead and intoxicated with DMN, did not show any signs of cell necrosis. There was an almost normal histological appearance except for a mild congestion (Fig. 2b). Under our experimental conditions, a significant inhibition of DMNdemethylase is observed at 24 h after lead administration; a lower activity, but statistically significant, still appears 48 and 72 h after lead administration. A complete recovery occurs at 5 days (Table III). Table IV shows the concentration of DMN in the plasma c o m p a r t m e n t of rats receiving DMN alone or after pretreatment with lead. Contrary to the
T A B L E II I N F L U E N C E BY L E A D N I T R A T E ON T H E L I V E R P O L Y S O M E S / T O T A L R I B O S O M E S R A T I O S A N D ON S E R U M A C T I V I T Y O F G L U T A M I C - P Y R U V I C T R A N S A M I N A S E IN D M N - T R E A T E D R A T S Treatment
Polysomes a
S-GPT b (milliunits/ml)
Total ribosomes (a) (b) (c) (d)
Control Lead DMN Lead + DMN
0.65 0.64 0.49 0.65
-+ 0 . 0 0 4 ± 0.028 ± 0.010 ~ 0.015
(4) (7) (4) (3)
30 41 1179 156
± 3 (7) ± 8 (7) ± 130 (7) ± 50 (6)
a M e a n + S.E. Statistical significance o f t h e d i f f e r e n c e s b y " t " test: a-c, c-d, P < 0 . 0 0 1 . R a t s were given lead n i t r a t e a t a dose o f 10~) pxnoles/kg b.w. 24 h b e f o r e D M N - i n t o x i c a t i o n a n d killed 90 rain a f t e r D M N a d m i n i s t r a t i o n . D M N was given at a dose o f 2 0 0 m g / k g b.w. T h e n u m b e r o f a n i m a l s is given in p a r e n t h e s e s . b M e a n +- S.E. Statistical significance o f t h e d i f f e r e n c e s b y " t " test: a-c, c-d, P < 0 . 0 0 1 ; a-d, P < 0 . 0 2 5 . R a t s were killed 24 h b e f o r e i n t o x i c a t i o n . Lead n i t r a t e was i n t r a v e n o u s l y i n j e c t e d at t h e dose o f 100 pxnoles/kg b.w. 24 h b e f o r e i n t o x i c a t i o n . D M N was intrap e r i t o n e a l l y i n j e c t e d a t a dose o f 1 0 0 m g / k g b.w. T h e n u m b e r o f a n i m a l s is given in parentheses.
111 B
A
0.~0
l
I
0 m
I
I
I
I
I
C
0.500
0
\ 2
4 L
Ef fluent (m,)
Fig. 1. Protection by lead nitrate against the polysomal disaggregation induced by DMN. Patterns of ribosome distribution in post-mitochondrial supernatants from livers of rats treated as follows: A, control untreated rat; B, rat given DMN (200 mg/kg b.w.) and killed 90 min after intoxication; C, rat given lead nitrate (100/~moles/kg b.w.) and killed 24 h after administration; D, rat given lead nitrate (100/,tmoles/kg b.w.) and 24 h later DMN (200 mg/kg b.w.); the rat was killed 90 rain after intoxication. The dashed lines separate the polysomes (to the right) from the monomeric-dimeric ribosomes (to the left). The relative areas are indicated as a percentage of the total ribosomal population. TABLE III DMN-DEMETHYLASE AT VARIOUS INTERVALS AFTER LEAD NITRATE ADMINISTRATION Rats received lead nitrate at the dose of 100/anoles/kg b.w., intravenously. The animals were killed at different intervals after administration, as stated in the table. For the enzymatic assay see under MATERIALS AND METHODS. Treatment
(a) (b) (c) (d) (e)
Control Lead Lead Lead Lead
Sacrifice (h)
HCHO formed a (nmoles/mg protein/30 min)
24 48 72 120
12.12 5.34 8.28 8.51 13.42
± 0.83 ± 0.78 ± 0.50 + 0.40 + 0.41
(4) (4) (4) (4) (4)
a Mean ± S.E. The number of animals is gi~ven in parentheses. Statistical significance of the differences by " t " test : a-b, P < 0.001 ; a-c, P < 0.01 ; a-d, P < 0.025.
i.a l...t b~
113 TABLE IV EFECT OF LEAD NITRATE ON THE DISAPPEARANCE OF INJECTED DMN FROM BLOOD All rats received a dose of DMN (500 mg/kg b.w.). Lead nitrate was given 24 h before DMN administration at a dose of 100 ~moles/kg b.w. Treatment
Time of sacrifice (after intoxication) (h)
DMN in blood a (g/ml)
DMN Lead + DMN DMN Lead + DMN DMN Lead + DMN
1 1 4 4 8 8
45.04 46.12 34.71 38.76 14.92 20.77
-+ 4.99 ± 0.79 -+ 1.48 + 2.87 ± 2.42 ± 1.65
(4) (4) (4) (4) (4) (4)
a Mean + S.E. Number of animals is given in parentheses. striking protective effect exerted by lead, the DMN clearance from plasma, even if lower at 4 and 8 h after DMN administration, does n o t seem to be influenced to a great e x t e n t by pretreating the animals with lead. DISCUSSION The toxic and carcinogenic effects induced by DMN have been attributed to metabolites which, arising f r om its biotransformation occurring within the drug-metabolizing e n z y m e system, react with cellular macromolecules (DNA, RNA, and protein) t hr ough alkylation processes [9] by an ultimate comp o u n d which is a m e t h y l carbonium ion [22]. Lead protects against DMN in the early as well as in the final stage of intoxication, t h a t is against polysomal disaggregation as well as against cell necrosis. Lead is a powerful inhibitor of the microsomal oxidative chain, with a long-lasting effect c om par e d with other chemicals [1]. Intoxication, such as CC14-poisoning, is prevented by pretreating the animals with lead [6]. CC14 is k n o w n to depend, for its toxicity, on the rate of its metabolism occurring in the drug-metabolizing e n z y m e system. McLean and Day [12] suggested for CC14 th at t he inhibition of the oxidative microsomal system, as obtained by feeding the rats with a protein-free diet, allows unaltered CCh to be expired f r o m the lungs. On the o t h e r hand, a p r o t e c t i o n by a protein-free diet against Fig. 2. Protection by lead nitrate against the morphological changes induced by DMN. Liver-sections taken from (a) a rat treated with DMN (100 mg/kg b.w.) and killed 24 h after intoxication and (b) a rat pretreated with lead nitrate (100/~moles/kg b.w.), then intoxicated with DMN (100 mg/b.w.) 24 h later; the rat was killed 24 h after intoxication. Staining: hematoxylin-eosin.
114
DMN-intoxication proceeds in spite of the fact that all DMN is eventually metabolised. Lead, as inhibitor of the drug-metabolizing enzyme system, can be compared to the protein-free diet, inasmuch as in both cases a protection occurs against two poisonings determined by chemicals, CCL4 and DMN, which require biotransformation as a basis of their toxicity. The clearance of DMN from the blood does not always parallel toxicity. Somogy et al. [23] observed a protection against DMN with pregnenolone16a-carbonitrile suggesting an alternative pathway by which DMN is metabolised into some harmless derivatives and this could be also the case for lead nitrate. This mechanism of protection differs from that possibly occurring by pretreating the animals with aminoacetonitrile, which increases DMN clearance from blood and, concomitantly, protects against liver toxicity [24]. Under our experimental conditions, a single dose of lead inhibits at 24 h the enzymatic activity of hepatic DMN-demethylase by 56%. Our data are in agreement with those of Scoppa et al. [5], who obtained, under the same experimental conditions, a decrease of other demethylases (e.g. aminopyrine and p-nitroanisole) between 50 and 65%, a decrease of cytochrome P-450 of 53% at 24 h, of 57% at 48 h, of 30% at 72 h, and 16% at 120 h. We are aware, from evaluating our results, of the difficulties in determining DMNdemethylase. This enzyme proceeds very slowly, as remarked by McLean and Day [10], at a rate of about 1/50 of the demethylation of aminopyrine. The unusual behaviour of DMN-demethylase, under different experimental conditions (e.g. after treatment with inducers of the drug-metabolizing enzyme system) points to the fact that the induction of the mixed-function oxidases is not always associated with the induction of DMN-demethylase, and, correspondingly, with increase in toxicity. Phenobarbital and 3-methylcholanthrene both inhibit DMN-demethylase, although inhibition by 3-methylcholanthrene is greater than that of phenobarbital [11,16,25]. 3-Methylcholanthrene has also been shown to protect rats against cell necrosis in another model of hepatotoxicity, CCl4-poisoning [26]. It seems so far that it is difficult to relate the inducibility of the drug-metabolizing enzyme system and liver toxicity by DMN. On the other hand, the inhibition of the drug-metabolizing enzyme system gives a clear picture of the relationship between drug metabolism and toxicity. Our data are in agreement with those of McLean and Verschuuren [12] and Pound et al. [14], who demonstrated that DMN toxicity is strongly decreased in animals with low activity of drug metabolizing enzymes. However, our data need further support as far as the metabolism of DMN is concerned, in order better to evaluate the action of lead on DMN-toxicity. ACKNOWLEDGEMENTS
This work was supported by "Consiglio Nazionale delle Ricerche", Roma, Italy.
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