Page 731 - The Toxicology of Fishes

Biomarkers 711

different aquatic organisms. However, as with the use of other biomarkers, histological alterations may
be influenced by factors other than chemical exposure. Given age, diet, environmental factors, seasonal
variation, and reproductive cycle, several structural states may represent normality and could be poten-
tially confounding issues in an attempt to use histological criteria as biomarkers. Regarding specific
alterations and references on histology of aquatic organisms, the reader is referred to Ashley (1975), Au
(2004), Couch and Fournie (1993), Ferguson (1989), Grizzle and Rogers (1976), Hinton et al. (1992),
Kubota et al. (1982), Myers and Fournie (2002), and Yasutake and Wales (1983).
Toxic effects on biochemical and physiological systems are ultimately expressed as changes in cellular
and subcellular morphology; thus, histopathological changes may be viewed as an integration of molecular
insults (Hinton and Lauren, 1990). Depending on the size of the fish, a number of important tissues may
be simultaneously studied while maintaining in situ cellular, tissue, and organ system relationships.
Maintenance of spatial relationships is possible and, in fact, required to appreciate biological effects
associated with toxicity in localized portions of an organ and the subsequent derangements in fluids,
tissues, or cells at other locations (Hinton, 1993). With small adults or fish larvae, proper orientation for
hemisection may yield samples through major viscera that can be viewed on only a few histological slides.
Such an approach enables examination of many potential targets of toxicant action and has been used
very effectively by a number of investigators. In medaka (Oryzias latipes) exposed to the estromimetic
chemical hexachlorocyclohexane, Wester and Canton (1986) evaluated morphological changes in gonads,
liver, kidney, pituitary, thyroid, spleen, and heart. The organs, or portions of organs, affected are recognized
targets of estrogen or are involved in the metabolism of products resulting from estrogenic responses.
Histopathologic analysis has proven to be a useful component in biomarker approaches to evaluate
the potential risk to aquatic organisms posed by exposure to both natural and anthropogenic chemicals
that may interfere with reproduction and development. In most fish, sex determination is under genetic
control; sex is determined by sex chromosomes after fertilization. In some fish, autosomes (pairs of
chromosomes with sex modifying genes) determine the sex, but this is less common (Jobling, 1995).
Sexual differentiation in fish is believed to be similar to mammalian systems whereby the presence or
absence of a testis-determining factor directs male or female differentiation. In teleosts, primordial germ
cells develop exterior to the gonadal region and then undergo migration to the gonad. The development
and positioning of the primordial germ cells are important markers of sexual differentiation, which is a
species- as well as sex-dependent process. In most cases, the differentiation of the gonad occurs in
females before males. This is usually determined by enumerating the number of primordial germ cells
entering or undergoing meiosis; for example, sexual differentiation in Japanese medaka occurs at
hatching, and in female medaka germ cells are in a proliferative state. Kanamori et al. (1985) reported
that sexual differences in the medaka were identifiable in the male medaka 10 days after fertilization.
The actual tubular structure of the testis was not seen until the fish was 15 to 20 mm in length, which
corresponds to the time when meiosis should occur (Kanamori et al., 1985). In some species, sexual
differentiation can be marked by the presence of an ovarian or testicular cavity. The evaluation of gonadal
development has been used as a biomarker response in field studies that have evaluated sexual determi-
nation and differentiation. Jobling et al. (1998), for example, demonstrated a high incidence of intersex-
uality in populations of roach (Rutilus rutilus) in the United Kingdom. The intersex condition was
characterized by the appearance of female characteristics in a typically male tissue and the progressive
disappearance of male characteristics. The investigators reported that the reproductive disturbances were
consistent with exposure to hormonally active substances and were associated with discharges from
sewage treatment works known to contain estromimetic chemicals.
For decades following the introduction of the concept of cellular pathology by Virchow, the under-
standing of morphological abnormalities at the cellular level was limited to data derived from fixed
images of cells and tissues as seen through the light microscope. Although the subjective nature of
conventional histopathology did not detract significantly from the discriminating power of this approach,
recent technological advances have improved resolution and identification of specific cellular and mac-
romolecular markers. Morphological changes in lysosomes, for example, have been employed as a
biomarker to evaluate toxic liver injury in various fish species (Kohler et al., 2002). Additionally,
technological advances in the field of histopathology have enabled increased quantitative assessment
using techniques such as digital optical imaging and immunohistochemical techniques.