Page 105 - The Toxicology of Fishes

Toxicokinetics in Fishes 85

transporters are plasma-membrane-bound export pumps; however, their amino acid sequence identity
and substrate specificity differ.
At least nine forms of the Mrp subfamily have been identified in humans. Some, such as MRP2,
MRP3, and MRP6 (capital letters indicate that these are human gene products), are primarily expressed
in the liver, kidney, and GIT. Others, such as MRP1, MRP4, MRP5, MRP7, and MRP8, occur in a
variety of tissues, including the prostate, testis, ovaries, pancreas, and lung, as well as the blood–brain
and blood–testis barriers. Most Mrps reside on the basolateral side of the plasma membrane, although
MRP2 has been localized to the apical side in epithelial tissues. Mrps actively transport organic anions
into bile and urine. Substrates for these transporters include xenobiotics conjugated with sulfate, glu-
tathione (GSH), and glucuronic acid. Several Mrps have also been shown to transport endogenous
molecules such as cyclic nucleotides, nucleoside analogs, and free GSH.
P-glycoprotein is an apical transporter of weakly cationic compounds and is expressed in a variety of
secretory cell types. Substrates for Pgp (also referred to as Mdr1) generally possess a molecular weight
between 350 and 1000, contain two planar rings, and exhibit a moderate degree of hydrophobicity.
Known substrates for Pgp include steroids, lipids, and peptides, as well as a variety of xenobiotics and
drugs such as adriamycin and vinblastin. Pgp appears to contribute to the regulation of some steroid
hormones. This regulation is achieved by the secretion of hormones from sites of synthesis, such as the
adrenal gland, and the modulation of hormone concentrations in target tissues such as the brain and testis.
A third class of transporters, organic anion transporter polypeptides (Oatps), are found in the liver, as
well as in other tissues. Members of the Oatp family reside on the sinusoidal (basolateral) membrane
of hepatocytes and transport negatively charged compounds. Unlike the Mrps and Pgp, Oatps function
as influx transporters.
Localization studies and functional experiments suggest that homologs to these transport proteins
operate in fish liver as well. Using immunohistochemical methods, homologs of mammalian Pgp have
been shown to exist in the bile canaliculi of several species (Curtis et al., 2000; Hemmer et al., 1995;
Kleinow et al., 2000). Studies with prototypical Pgp substrates have demonstrated the existence of an
ATP-dependent transporter function in both trout and channel catfish (Doi et al., 2001; Sturm et al.,
2001). Ballatori et al. (1999) used competitive inhibition methods as well as transport inhibitors to
demonstrate that uptake of lucifer yellow (a model organic anion) by skate hepatocytes is carrier
mediated. Additional studies with the skate suggest that glutathione conjugates are secreted into bile by
an efflux transporter with Mrp-like activity, located on the canalicular membrane (Rebbeor et al., 2000).
Fish excrete a wide variety of xenobiotics in the bile. A partial listing of these compounds includes
antibiotics (Samuelsen et al., 1995), insecticides (Lech et al., 1973), dyes (Plakas et al., 1992a), herbicides
(Schlenk and Moore, 1993), polyaromatic hydrocarbons (Collier and Varanasi, 1991), metals (Grosell
et al., 2001), and natural products (Sahin et al., 1996). Early studies with rainbow trout showed that
chemical concentrations in bile may be much higher than those in the surrounding water (Lech et al.,
1973; Statham et al., 1976). Guarino and Lech (1986) summarized the results of studies with fish exposed
to more than 40 drugs and chemicals of varying molecular weight. The data were examined to determine
bile/plasma concentration ratios and the percentage of compound eliminated in bile or urine. Bile/plasma
concentration ratios were greater than 1 for over 85% of compounds examined, suggesting that fish were
capable of concentrating xenobiotics in bile relative to plasma. The relative importance of biliary
excretion as a route of elimination increased with molecular weight, although other factors such as the
charge on the molecule were important. Generally, chemicals that are highly polar and have molecular
weights greater than 600 are excreted largely in bile (Gingerich et al., 1977; Schmidt and Weber, 1973).
Compounds of intermediate molecular weight (300 to 600) and polarity may be eliminated in bile as
well as by other routes (Allen et al., 1979; Hunn and Allen, 1975; Kobayashi et al., 1977). From these
limited datasets we may conclude that the same criteria (molecular weight and polarity) that apply to
biliary excretion in mammals are operative in fish.

Metabolic and Physiological Impacts
Biotransformation may substantially increase biliary elimination of xenobiotics by increasing their
solubility and creating conjugated compounds that are substrates for active transport systems. Stehly
and Hayton (1989c) examined the metabolism and biliary excretion of pentachlorophenol in several fish