|Abstract or Summary
- In rodent models as well as in fish models there are significant
species differences in the susceptibility toward aflatoxin B₁ (AFB₁)
carcinogenesis. Mouse is less susceptible toward AFB₁ carcinogenesis
than rat. Researchers have come to the conclusion that the lower
susceptibility of mice is not a result of less effective activation of AFB₁,
but rather of more effective inactivation of the toxic intermediate
AFB₁-2,3-epoxide, and especially inactivation through the glutathione
(GSH) conjugation of the epoxide.
Rainbow trout fed Oregon Test Diet are more sensitive toward AFB₁
hepatocarcinogenesis than coho salmon, or, than trout fed the inhibitors
β-naphthoflavone (BNF), 1ndole-3-carbinol (I3C), or Aroclor 1254
(PCB). This study examined the role of AFB1-glutathione (AFB₁-SG) conjugation In these differences.
A tritiated AFB₁-glutathione conjugate standard (³H-AFB₁-S6) was
produced in vitro using mouse liver S-9 fraction as a source of GSH
transferase. It was purified by reverse phase HPLC, and its structure
verified by amino acid analysis and mass spectrometry.
Coho salmon and rainbow trout fed the various diets were injected
i.p. with ³H-AFB₁ (49μCi, 50 μg/ kg fish); bile, liver and kidney were
collected at 24 h. Recovery of total aflatoxin radioactivity was determined
for all three tissues, and the hepatic AFB₁-DNA binding was also
determined. Bile metabolites were quantitated by reverse phase HPLC
using the mouse ³H-AFB₁-SG as a standard.
The resistance of coho salmon toward AFB₁ carcinogenicity was
supported by a 20-fold lower hepatic AFB₁-DNA binding compared to
control trout. AFB₁-SG was detected in bile only in control, BNF, and I3C
fed trout, at < 1% of total recovered metabolites, and at < 0.2% of the
original dose, being highest in control trout. The major conjugates were
glucuronides of aflatoxicol (AFL) and aflatoxicol-M₁ (AFL-M₁) (80-902
of the total recovered metabolites).
In vitro metabolism studies using isolated liver cell fractions
supported the in vivo metabolism results. Less than 0.531 of the original
AFB₁ dose was converted to AFB₁-SG conjugate in salmon and trout samples. In contrast, with isolated mouse liver cell fractions
approximately 25% of the original AFB₁ dose was conjugated with GSH.
The GSH concentration of control trout liver was 2.9 mmol/ kg.
Coho salmon had GSH concentration 70% of that found in control trout. In
BNF pre-fed trout liver GSH concentration was enhanced by 25% compared
to controls. Liver GSH transferase activity using l-chloro-2,4-
dlnitrobenzene as substrate was 1.15 μmol/min/mg protein in control
trout. This enzyme activity In salmon was only 26% of that found in control
trout. A 62% elevation In GSH transferase activity compared to controls
was detected in trout fed BNF diet. There is no apparent correlation
between liver GSH or GSH transferase activities among the various groups,
and their relative sensitivities to AFB₁ carcinogenesis.
This study indicates that AFB₁-SG conjugation is not a significant
pathway in salmon and trout fed control diets, or trout fed various
inhibitors, and cannot account for the variation In AFB₁ sensitivity.