| dc.description.abstract | This thesis reports the results of a study designed to
compare the effect of different hepatocarcinogens - N,N-dimethyl-
4-phenylazoaniline (DAB) and diethylnitrosamine (DEN) -
on the activities of representative microsomal and lysosomal
enzymes involved in the metabolism of exogenous chemicals
throughout the entire carcinogenic process in adult male rats.
1) The effect of feeding a low protein diet containing
0.06 % N,N-dimethyl-4-phenylazoaniline for 29 weeks on the
activity of DAB-azoreductase, nitroreductase (p-nitrobenzoic
acid as substrate), N-oxidase (N,N-dimethylaniline as substrate),
N-demethylase (DAB as substrate), cytochrome P-450,
NADPH-cytochrome c reductase, beta-glucuronidase and arylsulphatase
were studied. Rapid decreases occured in the activity
of the first 6 enzymes, reaching minimum values between
4 and 8 weeks. Activities then increased in all cases
to control or nearly control levels. This rate of increase
was least for cytochrome P-450. At 4 weeks azoreductase activi
ty wi th 2- (4'-di (2"- bromopropyl )aminophenylazo )bf:llzoic
acid (CBlO-252) as substrate was actually higher than in control
rats. Early increases occured in the activities of betaglucuronidase
and arylsulphatase A and the latter never dropped
below the control level.
An investigation was made of the differential effects
of the dye feeding on some of the enzyme activities in the
two major lobes and differences were found.
The effect of phenobarbitone pretreatment on the DABfed
rats was studied at 4 weeks interval. The activity of
DAB-azoreductase and nitroreductase were increased throughII
out the whole period, while the activities of the lysosomal
enzymes.were decreased.
After feeding DAB for ~.weeks the effect of phenobarbitone
and 3-methylcholanthrene on the activities of_DAB-azoreductase,
CBlO-252-azoreductase and components of the azoreductases
- cytochrome P-450, NADPH-cytochrome c reductase,
the carbon monoxide-sensitive pathway and the NADH-dependent
pathway were studied. The activity of CBlO-252-azo-
reductase was not induced by.phenobarbitone or 3-methylcholanthrene,
and CO did not inhibit the reduction. Its reduction
was dependent only slightly on NADH. CO caused a greater
relative 'decrease in the ac tIvi ty of DAB-azoreductase
in dye-fed anim&ls following phenobarbitone &nd 3-methylcholanthrene
pretreatment, implying a greater role of cytochrome
P-450 in dye-fed rats.
2) The effect of feeding DEN (qiethylnitrosamine) by
stomach tube for 32 weeks on the activities of DAB-azoreductase,
nitroreductase, NADPH-cytochrome c reductase, cytochrome
P-450, CBlO-252-azoreductase, beta-glucuronidase
and arylsulphatase A was studied. Decreases occured in the
activities of the first 4 enzymes reaching a minimum between
4 and 7 weeks, similar to the DAB-feeding. The activities
returned to normal or nearly normal level before decreases
in the activities were observed at the end of the
experiments. The activity of CBlO-252-azoreductase showed
increased level after 4 weeks and remained above the control
level throughout the experiment. Increase in the activity
of the lysosomal enzymes in the "lysosomal!! as well as the
"microsomal" fractions was observed, the increase being deIII
layed in the microsomal fraction.
The effect of phenobarbitone pretreatment on the activities
of the enzymes in the diethylnitrosamine-fed rats
was studied at 4 weeks interval. The activities of the lysosomal
enzymes showed decreased levels, whereas DAB-azoreductase,
cytochrome P-450 and NADPH-cytochrome c reductase
all showed slightly increased activity.
3) The effect of experimental cirrhosis and hepatitis ,;
on the activities of some of the enzymes was investigated.
The activities of the drug metabolizing enzymes showed a
decrease~in both pathological states, whereas the activity
of the lysosomal enzymes was increased. CBIO-252-azoreductase
showed increased activity in hepatitis and decreased
activity in cirrhosis.
4) The effect of a single large dose of a liver carcinogen
- N,N-dimethyl-4-phenylazoaniline, urethane and 2-acetylaminoflourene
- on the activities of DAB- and CBIO-252
azoreductase, NADPH-cytochrome c reductase and the level of
cytochrome P-450 has been studied.
5) It has been demonstrated, "that the system involved
in the reduction of 2-(4'-di(2"-bromopropyl)aminophenylazo)
benzoic acid (CBIO-252) is localized mainly in the 108000 x
g supernatant fraction of the rat liver homogenate, and that
it is also present in other organs, particularly in the spleen.
N,N-dimethyl-4-phenylazoaniline (DAB)-azoreductase is present
almost entirely in the microsomes and high activity is
found only in the liver. The pH maxima for CBIO-252-azoreductase
and DAB-azoreductase were 6.2 and 6.9, respectively.
Methylred-azoreductase had in most respects properties simiIV
lar to CBIO-252-azoreductase.
The use of enzyme inhibitors and other additives showed
that CBIO-252-azoreductase was not identical with xanthine
oxidase or dihydrofolate reductase. Its activity was not
affected by carbon monoxide, phenobarbitone or 3/methylcholanthrene
pretreatment. Enhancement of the activity by ferrous
ions and flavin-adenine-dinucleotide indicates, that_at
least part of the reduction system could involve a flavoprotein
with FAD as the prosthetic group.
CBIO-252- and methylred-azoreductases were inhibited
by menadione (vitamin K
), cyanide ion and propylgallate.
A diaphorase pre~aration from pig heart was able to
reduce both CBIO-252 and methylred with a NADPH-generating
system as well as a NADH-gener.s.ting system. The properties
listed above and dependence of enzyme activitity on NADPH
and NADH indicate a similarity to DT-diaphorase (NAD(P)H
dehydrogenase).
Non-enzymatic reduction of CBIO-252 by reduced glutathione
and ascorbic acid was also demonstrated.
The involvement of different enzyme systems in the reduction
of N,N-dimethyl-4-phenylazoniline on the one hand
and 2-(4'-di(2"-bromopropyl)aminophenylazo)benzoic acid on
the other, are suggested to be due to the presence of the
carboxylic group in the latter compound. | en |
