IN VITRO HEPATIC METABOLISM OF THE PHYTOBIOTIC 1,8- CINEOLE IN DOMESTIC FOWL
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Date
2018-08-13
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COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD
Abstract
Chickens reared under intensive systems are likely to be exposed to feed
additives and various xenobiotics. With the ban of antibiotics as in-feed growth
promoters (AGPs), there is requirement of alternative method to improve the bird’s
performance capacity and also cope well with harsh conditions during rearing period.
Phytobiotics, especially essential oils (EOs), are a new class of feed additives that have
attracted attention due to their attributed antimicrobial and growth promoter properties.
1,8-cineole is a naturally occurring monocyclic monoterpene ether with an
aromatic and camphor like odour and is one of the essential oil (EO) components used
in poultry feed as phytobiotic. Even though the metabolism of 1,8-cineole is well
studied in rats, rabbits, koala and brush tail possum and humans, its fate in poultry is
not yet reported. Cytochrome P450 enzymes (CYP) are a group of monooxygenases
playing a significant role in the biotransformation of several kinds of xenobiotics.
CYP3A enzymes have been reported as responsible for metabolism of 1,8-cineole in
rat and human liver microsomes. Avian and other mammalian species have distinctly
different potential capacities of metabolizing drugs. The difference in the metabolic
pathway and metabolic enzymes involved in biotransformation of drugs is one of the
factors contributing for species variability in pharmacological responses.
Although mammalian and avian CYPs are not strictly orthologs, some
substrates were found to work for both groups. Hence, the present study was conducted
in vitro to identify the cytochrome P450 enzyme orthologs involved in the
biotransformation of 1,8-cineole in chicken hepatic S9 and microsomal fractions. The
approaches used included the use of specific cytochrome P450 (CYP3A4) inhibitors
and the correlation of prototype substrate activities with the formation of the
hydroxylated metabolite of 1,8-cineole. Nifedipine and phenacetin were used as
specific substrates for the CYP3A and CYP1A isoforms and ketoconazole was used as
an inhibitor.
Twelve day old Gramasree layer chicks were purchased from hatchery unit,
Instructional Livestock Farm Complex, College of Veterinary and Animal Sciences,
Pookode and were reared under standard management practices for 12 weeks. The
chicks were fed with standard layer feed diet for starter (0-8 weeks) and grower (8-12
weeks) as per Bureau of Indian Standards, 2007 and had free access to water ad libitum
until the experiment.
A high performance liquid chromatography method was validated and applied
for the determination of nifedipine, phenacetin and their formed metabolites nifedipine
oxide and acetaminophen in hepatic microsomes and S9 fractions. Determination of
nifedipine and nifedipine oxide were carried out at a flow rate of 1 mL/min using the
mobile phase consisting of methanol and water in the ratio of 65:35. Phenacetin and
its metabolite acetaminophen were determined at a flow rate of 1 mL/min by using the
mobile phases consisting of methanol: water (70:30) and water: acetonitrile (85:15)
respectively. Analysis of 1,8-cineole and its metabolite was done by gas
chromatography mass spectrophotometry(GC-MS). The extraction efficiency was also
determined for nifedipine, nifedipine oxide, phenacetin, acetaminophen and 1,8-
cineole by comparing the peak areas from drug free samples spiked with known
quantities of drug in the range of concentration of calibration curves and standard
solutions with suitable solvent in which it is soluble, injected directly into analytical
column.
Chickens of ninety days age were euthanized by complete cranial decapitation
followed by exsanguination. The liver was immediately collected and washed with icecold 1.15 per cent of potassium chloride solution. Both the body and liver weight were
recorded. A portion of the collected livers (major lobe) was processed at 4 ºC for the
generation of S9 and microsomal fractions. In vitro incubation studies were carried out
in S9 and for with and without NADPH generating system with nifedipine, phenacetin
and 1,8-cineole in presence and absence of the inhibitor ketoconazole at predetermined
time intervals. The enzyme activities for the CYP isoforms were correlated with the
formation of the metabolites. Analysis of various drug metabolisms were done using
Graphpad prism 5 software and nonlinear regression analysis of drug disappearance
versus time was done with mycurvefit.
No mortality of the birds was recorded when reared strictly under intensive
system. The birds at the end of 12th week attained an average body weight of. 771.33 ±
64.96 g. The nifedipine and its metabolite were best separated with retention times of
8.02 and 6.01 min respectively. The drug phenacetin and its metabolite had a retention
time of 4.2 and 5.5 min respectively. Ketoconazole was detected at 220 nm with a
retention time of 1.9 minute using the mobile phase mixture of methanol: 0.1% formic
acid (90:10, v/v). The retention time of 1,8-cineole and its metabolite were 7.9 and
13.1 min respectively.
An average obtained liver weight of 19.79 ± 2.88 g was obtained which was
approximately 2.56 per cent of the average body weight for Gramasree birds. The
microsomal protein in the present study was 28.42± 0.780 per gram of fresh weight
tissue. Incubation of 1,8-cineole in the hepatic S9 fraction with a protein concentration
of 7 mg/ml up to 180 min did not exhibit any significant decrease in the concentration
of the parent compound compared to samples at ‘zero’ min. On the contrary, 1,8
cineole was significantly metabolized by the microsomal fraction with a linear decrease
in the concentration of the drug. This could be due to the difference in the metabolic
enzymes present in the cytosolic and microsomal fractions. No significant difference
in metabolic activity was noted between the male and female birds. The formation of
nifedipine oxide at different incubation times indicated the existence of chicken CYP
ortholog of the human enzyme studied. Phenacetin, also metabolized by CYP1A2
isoenzyme was used as a specific substrate probe for determining its activity.
Phenacetin was not metabolized by the hepatic cytosolic enzymes till 180 minutes.
However, both phenacetin and 1,8-cineole was metabolised in the S9 fraction 6 hours
post incubation indicating low CYP activity in the hepatic S9 fraction. .
The study indicated that CYP3A isoform catalysed the hydroxylation of 1,8-
cineole in chicken liver microsomes with the formation of 2α-hydroxy-1,8-cineole. The
human isoenzyme CYP3A specific inhibitor ketoconazole significantly inhibited the
metabolisms of nifedipine and 1,8-cineole in chicken liver microsomes. Since
ketoconazole is also reported as a nonspecific inhibitor of CYP 1A in chickens, the role
of CYP 1A2 in the metabolism of 1,8-cineole in chicken in the present study cannot be
ruled out. Further, the rate of 1,8- cineole biotransformation is low in chickens when
compared to biotransformation in humans and rats in vitro.