Page 589 - The Toxicology of Fishes

Chemical Carcinogenesis in Fishes 569

Importantly, the responses of fish to their environment integrate the processes of uptake, distribution,
bioavailability, metabolism, degradation, and the combinatorial effects of complex mixtures (Long and
Buchman, 1990).

Temperature
Fishes are poikilothermic, with body temperatures that fluctuate with ambient temperatures. Because
they are poikilotherms, it has been possible to analyze the effect that changes in temperature exert on
carcinogenesis in medaka (Kyono, 1978; Kyono and Egami, 1977; Kyono et al., 1979; Kyono-Hamagu-
chi, 1984). Following exposure to DEN for 6 to 8 weeks at 25°C, liver tumors appeared after 11 to 13
weeks. The greatest effect on tumorigenesis was seen when fish were initiated and allowed to grow out
at high temperature (25°C). When the initiation temperature was reduced to 5 to 8°C, this resulted in
no tumors; however, for grow out after initiation at a low temperature (25°C), a few tumors resulted.
Labeling and mitotic indices were performed on the above groups, and the results showed the highest
rate of DNA synthesis and cell divisions at high initiation and high grow-out temperatures.
Similar results were seen in trout, and additional mechanisms were investigated (Curtis et al., 1995).
First, trout were acclimated at one of three temperatures—cool (11.0°C), warm (18.0°C), or intermediate
(14.5°C)—for 1 month. Next, they were exposed to AFB for 30 minutes and then reared for 9 months.
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Tumor incidence and multiplicity tracked increasing temperature. In addition, downward temperature
shifts reduced AFB –DNA adduction. In a follow-up study (Zhang et al., 1992), trout were acclimated
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at one of three temperatures and then immersed in [ H]-AFB at their respective acclimation temperatures
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or at higher temperatures. The total radioactivity was used to establish uptake. DNA adducts were less
persistent in fish maintained at 21 days at 18°C than at 10°C. These findings suggest that temperature
modulation of AFB genotoxicity occurs by three mechanisms: uptake by liver and the formation and
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persistence of resultant DNA adducts (Zhang et al., 1992). In the most recent trout temperature studies
(El-Zahr et al., 2002), the PAH DMBA was administered in a single exposure (20-hour duration) to trout
previously acclimated at 10, 14, or 18°C. Various temperature shifts were used to assess the effects on
hepatic uptake, DNA adducts, and eventual tumor incidence. When fish were shifted to higher temperature
after a 4-hour DMBA exposure, DMBA in liver increased with temperature. In fish exposed to 14°C
and then shifted to 10°C or to 18°C for 3 days, adduct levels were higher in the former. After 21 days,
adduct persistence was less in fish at 18°C than those at 10°C. After 9 months’ rearing, the incidence
of stomach, liver, and swim bladder tumors showed a dramatic increase with increased rearing temper-
ature. The investigators considered the loss of persistence of DNA adducts with higher temperatures to
be evidence for increased error-prone DNA repair at warmer temperature (El-Zahr et al., 2002).

Crowding/Stocking Density
One of the most important advantages of aquarium fishes such as medaka is that large numbers of
animals can be reared in small indoor aquaria; however, no standardized protocol exists that provides
stocking density guidelines for carcinogenesis studies, and, indeed, stocking rates are frequently not
stated in published reports. Stocking density, which has been shown to have significant effects on growth
in other fish species (Canario et al., 1998; Chua and Teng, 1979), was reported infrequently in carcino-
genesis studies published between 1982 and 1999 utilizing medaka as test animals (Davis et al., 2002).
Stocking densities commonly used in carcinogenesis assays can significantly affect growth rate and
fecundity in medaka (Davis et al., 2002). In light of the interrelationships of food restriction (Avula et
al., 1999; Zhu et al., 1999) and steroid hormones (Degen and Metzler 1987; Virna et al., 1996) with
cancer, stocking density, with its effect on growth and reproduction, can potentially impact carcinogenesis
study outcomes. As an added factor, stocking density can impact water quality, depending on the type
of aquarium system used. Significant reductions in dissolved oxygen and increases in total ammonia
nitrogen have been reported with increasing the stocking density of palmetto bass kept in aerated outdoor
ponds (Liu et al., 1999). Also, body weight and daily egg production were found to be further reduced
when, with stocking density held constant, the feeding rate was reduced from excessive feeding (20%
of body weight daily up to 8 weeks of age, then 15% of body weight daily thereafter) to mildly restrictive
(10 to 12% of body weight daily up to 6 weeks of age, then 5% of body weight daily thereafter) (Davis