Dimethylbenz (a) anthracene (EMBA), a carcinogen that requires
metabolic activation to produce active metabolites capable of
binding to DNA, has been studied in the trout and other fish.
Polycyclic aromatic hydrocarbons (PAH) are of importance as they
are ubiquitous in the environment and their carcinogenic effects
in fish from contaminated waters are an important indication of
the pollution risks to man. Since such pollution risk assessment
presents the involvement of multiple agents, the study of the
modulation of PAH-DNA binding produced by other agents is
important. In this study the effect, of dietary pretreatment at
500 ppm, 100 ppm and 2000 ppn, using BNF, Aroclor 1254, or
indole-3-carbinol (I3C) respectively on DMBA-DNA binding was
examined. To study the effect of age on sensitivity to DMBA-DNA
binding, adult trout and fry were used in two separate studies.
The fish were fed treatment diet for at least two weeks. Fry were then injected with [³H] DMBA, at 22.4 μCi/3.9 x 10⁻² μmole/fish and adult trout at 284 μCi/1.58 μmole/fish. Liver DNA
was isolated, purified and binding of radioactivity to DNA was
examined and computed as the covalent binding index (CBI). Mean
CBI for control dietary group vising adult trout was 1000 fold
lower than for fry. Statistical analysis of covalent binding
index for the treatment groups revealed that a statistically
significant (p < 0.05) inhibition in DNA-DMBA binding response
in adult trout and fry was produced fcy the DNF dietary treatment
only.
Diethylnitrosamine (DENA), a potent hepatocarcinogen in
several animal species belongs also to the class of compounds
that require metabolic activation. Dietary treatment and
continuous exposure of trout to the carcinogen in water, have
produced hepatocellular carcinctnas. The water exposure also
produced a dose related DNA ethylation of the O⁶ position of
guanine, believed to be the promutagenic adduct produced after
DENA exposure. This study examines two other routes of exposure
to DENA, in vitro hepatocyte incubations and i.p. injection.
Adult trout and fry were injected with [³H] DENA. Adult fish
received 3.3, 16.5, and 33 mg/kg DENA, and fry received 10, 50
and 100 mg/kg. Hepatocyte incubation was performed with doses up
to 220 μM [³H] DENA, or 1 mM unlabelled DENA. DNA from fish
livers and from hepatocyte pellets was isolated, purified and
examined for radioactive binding of the DENA metabolites or in
the case of the unlabelled DENA, was analyzed for O⁶ and N7
adduct using an HPIC technique with fluorescence detection. O⁶-EtG adduct after DENA exposure, in DNA of hepatocytes obtained
from trout pretreated with beta-naphthoflavone (BNF, a known
inducer of cytodhrcme P-450 dependent enzyme activities involved
in the activation of xenobiotics) was below the limits of
detection of the HPDC-fluorescenoe detection procedure used. To
examine further if the lack of DNA binding and absence of the
O⁶-EtG adduct was due to rapid repair, the persistence of O⁶-EtG
after exposure to 40 nM ethylnitrosourea (ENU, a direct
ethylating agent) was studied in hepatocytes at 2, 4, and 5
hours after treatment. The activity of the alkyltransferase
protein involved in the repair of alkylguanines also was
determined using liver extracts from adult rainbow trout. The
studies did not reveal a significantly high rate of repair. It
is concluded that i.p. injection and in vitro hepatocyte
incubations are not a good method for studying the kinetics of
activation and DNA binding of DENA in the rainbow trout. The
i.p. route may lead to substantial loss of the dose of the
carcinogen administered and hepatocyte incubations are limited
by the toxic effects of increasing carcinogen concentration. The
reasons mentioned above, coupled with low levels of metabolism
of nitrosamines in trout results in the inability to detect and
study the appearance, persistence and repair of the O⁶-EtG
adduct. / Graduation date: 1989
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/27201 |
| Date | 11 August 1988 |
| Creators | Van Winkle, Samina |
| Contributors | Bailey, George S. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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