Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons (PAH) are widely distributed in the environment and probably cause neoplasms in wild fish (Baumann, 1989; Baumann and Harshbarger, 1995). The PAH carcinogens consist of 2-6 fused benzene rings with or without alkyl substitutions. Members of this group include 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 20-methylcholanthrene (formerly termed 3-methylcholanthrene), and several compounds with nitrogen or sulphur atoms included in their rings. Polycyclic aromatic hydrocarbons are hydro-phobic and therefore prone to accumulate in sediments (McElroy et al., 1989).

Major sources of PAH include crude oil and products produced during burning of fossil fuels or organic matter (Tan and Heit, 1981), and most PAH are delivered to aquatic environments by atmospheric deposition or through runoff (Hoffman et al., 1984; Prahl et al., 1984). Levels of PAH in aquatic sediments can be chronologically correlated to the advent of the Industrial Revolution (Gschwend and Hites, 1981), and there are examples of locally high levels of PAH related to industrial sources such as creosote plants (Bieri et al., 1986; Krahn et al, 1986).

Although PAH are degraded by some fungi and bacteria under aerobic conditions (Cerniglia and Heitkamp, 1989), PAH tend to accumulate in sediments and in some aquatic animals. Fish and shrimp can efficiently metabolize and excrete PAH; therefore, less accumulation of PAH occurs than in bivalves and gastropods, which metabolize PAH slowly and so are subject to accumulation of large tissue burdens (Neff et al., 1976; Roesijadi et al., 1978; Varanasi et al., 1985). In a Puget Sound study, English sole (Pleuronectes [=Parophrys] vetulus) were found to have liver concentrations of benzo-[a]pyrene that were below detection limits (<25 ng g-1 dry weight), while their stomach contents (annelids, mollusks, crustaceans, and echinoderms) had 570 ng g-1 dry weight, and sediments in the collection area had from 170 to 550 ng benzo[a]pyrene g-1 dry sediment. None of the 25 hydrocarbons quantified were present in fish liver in higher concentrations than levels in stomach contents or sediment (Malins et al., 1985).

Metabolism of PAH by fish has been extensively studied (Varanasi et al., 1989). Fish metabolize PAH to form unstable intermediates that can form DNA adducts, but there is still uncertainty regarding the relation between PAH adduct formation and neoplasia. Common carp have a much lower neoplasm frequency than brown bullheads in environments with high PAH levels (Brown et al., 1973), but contrary to expectations, common carp make more PAH-related DNA adducts than do brown bullheads (Steward et al., 1989; Sikka et al., 1990). Obviously the relation between adduct formation and neoplasia is complex.

Field studies

Several epizootics of neoplasia in fish appear to be related to PAH contamination. However, most of these cases involve complex mixtures of chemicals and the contribution of a single carcinogen to the overall incidence of neoplasia is difficult to discern. The following studies strongly implicate PAH as a cause of neoplasia in certain populations of wild fish.

The Elizabeth River runs through a heavily industrialized area of Virginia and is highly contaminated with PAH (Bieri et al., 1986). Mummichogs (Fundulus heteroclitus) from a portion of the river that had up to 3900 mg PAH kg-1 of sediment (adjacent to an abandoned creosote plant and an active oil transfer and storage site) had papilloma, schwannoma and haemangio-endothelioma (Hargis et al., 1989). The overall prevalence of neoplasms was 2%. Mummichogs in later collections in the Elizabeth River at a site with 2200 mg PAH kg-1 of sediment had a 73.3% prevalence of hepatic foci of alteration and a 35% prevalence of hepatocellular neoplasms (Vogelbein et al., 1990). Only 600 m away and across the river, PAH concentration was 61 mg kg-1 sediment, and mummichogs from this area had no hepatic lesions. Mummichogs from the contaminated site also had neoplasms of the exocrine pancreas (Fournie and Vogelbein, 1994).

The Niagara River area near Buffalo, New York, has several sites that contain high concentrations of PAH (Black, 1983). Neoplasms of fish from this area included dermal neoplasms in freshwater drum (Aplodinotus grunniens) and oral papillomas in white suckers. Freshwater drum had dermal neoplasms with frequencies as high as 16.7% in Lake Erie near Wanakah, New York, and 13.3% at the confluence of Frenchmans Creek and the Niagara River. The neoplasms of freshwater drum were more common in larger fish. White suckers over 30 cm long had oral papillomas with an overall frequency of 8.5%. Although a high prevalence of neoplasms was observed at some locations with relatively low concentrations of PAH in sediment, freshwater drum and white suckers can move freely from areas of high sediment concentration of PAH to nearby areas with low concentration. Various types of neoplasms were found in five additional species of fish, including a 17% prevalence of grossly visible skin or liver neoplasms in large adult brown bullheads in the Buffalo River, New York (Black, 1983; Black et al, 1985a).

The Black River drains a heavily industrialized portion of northern Ohio, and its sediments are heavily contaminated with PAH (West et al., 1988). Ten types of PAH were identified in brown bullheads from the Black River, and concentrations of these PAH were much higher than in reference fish (Baumann et al., 1987). Brown bullhead tissues from this area contained 3.1 mg kg-1 of phenanthrene plus lower levels of other PAH. There was also 1300 ng PCB g-1 wet weight in Black River fish, compared with 50 ng g-1 in reference fish.

Brown bullheads collected from the Black River had a high prevalence of liver, skin and lip neoplasms (Baumann et al., 1987, 1990). Most liver neoplasms in brown bullheads were cholangiocarcinomas, approximately 60% of the skin and lip neoplasms were papillomas, and the remaining skin and lip tumours were squamous cell carcinomas. No evidence of viruses in the lesions was found with electron microscopy. Prevalence of neoplasms in these fish was age dependent. Skin and lip neoplasms occurred in less than 1% of 2-year-olds, but frequencies in age-4 fish were as high as 32% for lip neoplasms and 18% for skin neoplasms. Prevalence of liver tumours was less than 2% in 2-year-olds, exceeded 11% in 3-year-olds, and was 28-44% in age-4 fish. Prevalence was even higher in 4-year-olds sampled in September (54%) and in 5-year-old fish (60%), but few fish survived to this age. Brown bullheads collected from two reference sites had no liver neoplasms, but there was a 1.5% frequency of lip tumours in 3-year-olds at one site. The cause-and-effect relationship between PAH exposure and hepatic neoplasms was strengthened by the decline in prevalence of hepatic neoplasms in brown bullheads after PAH levels decreased following closure of a coking facility in 1983 (Baumann and Harshbarger, 1995).

Puget Sound is perhaps the best characterized site of a PAH-associated epizootic of fish neoplasms (Malins et al., 1987, 1988; Myers et al., 1990, 1991; Stein et al., 1990). Although a few areas in Puget Sound have sediments with high concentrations of anthropogenic chemicals, most of Puget Sound is less polluted, allowing comparisons of fish collected from locations with different levels of sediment contamination. Over 900 different organic compounds were identified in sediments of Commencement Bay (Malins et al., 1984a). Aromatic hydrocarbons were also found in invertebrate animals recovered from the stomach of English sole from Puget Sound (Malins et al., 1985), indicating that organic chemicals present in sediment are available through the diet. There were positive correlations between prevalence of hepatic neoplasms in English sole from several locations and sediment concentrations of PAH and metals (Malins et al., 1984b). However, there was a higher correlation between concentration of bile metabolites of aromatic compounds and prevalence of hepatic neoplasms (Krahn et al., 1986).

Prevalences of hepatic neoplasms in English sole from polluted areas of Puget Sound vary from 2.6 to 32% depending on collection site (Pierce et al., 1978; Malins et al., 1984b, 1985; Becker et al., 1987; Myers et al, 1987). No fish with neoplasms were found in several minimally polluted locations. Site of capture and fish age were the most important risk factors for neoplasms as well as for other hepatic lesions (Rhodes et al., 1987). Myers et al. (1987, 1998) found that certain types of non-neoplastic lesions in the liver of English sole had high frequencies of co-occurrence with hepatic neoplasms and were useful indicators of exposure to carcinogens.

The starry flounder inhabits the same areas of Puget Sound as the English sole and has similar feeding habits but a much lower prevalence of neoplasia (Pierce et al., 1980). This difference is caused by quicker conversion of PAH to proximate carcinogens and slower detoxification of reactive intermediates by English sole (Collier et al., 1992).

Laboratory studies

Bath treatments with PAH are effective in producing neoplasms in fish. Multiple exposures of Poeciliopsis spp. to a 5 mg l-1 aqueous suspension of 7,12-dimethylbenz[ a] anthracene at weekly intervals produced hepatic neoplasms with a frequency of nearly 50% at 7-8 months (Schultz and Schultz, 1982). These hepatocellular carcinomas ranged from well differentiated trabecular forms to anaplastic types with deeply basophilic, pleomorphic, spindle-shaped cells. This experiment also produced several highly invasive lymphosarcomas. Hawkins et al. (1988b) exposed guppies and medaka to water containing 250 mg l-1 benzo[a]pyrene in a bath treatment. Both guppies and medaka developed invasive, polymorphic, trabecular hepatocellular carcinomas with high mitotic rates, but there was a higher neoplasm frequency in medaka than in guppies at all sampling intervals. Hepatic neoplasms were the most common tumours in guppies exposed for 6 hours once weekly for 4 weeks to 7,12-dimethylbenz-[a]anthracene, but low numbers of rhabdomyosarcoma, renal adenocarcinoma, neurilemoma and undifferentiated sarcoma also occurred (Hawkins et al., 1989).

Rainbow trout fed benzo[a]pyrene continuously for 18 months had a 25% frequency of hepatocellular carcinoma; neoplasms were first noted at 12 months (Hendricks et al., 1985). In the same study, 50 fish received monthly intraperitoneal injections of benzo[a]pyrene; 25 fish developed invasive hepato-cellular carcinomas, one fish developed hepatic fibrosarcoma, and one fish developed a papillary adenoma of the swimbladder. Initial attempts to demonstrate that PAH were carcinogenic when injected into rainbow trout embryos resulted in low numbers of hepatic neoplasms and high mortality (Metcalfe and Sonstegard, 1984; Black et al., 1985b). After embryos were bathed in 7,12-dimethylbenz[a]anthracene, about 80% of the fish developed hepatic neoplasms; gastric adenomas and nephroblastomas also occurred (Fong et al, 1993).

An organic fraction extracted from sediment of the Black River, Ohio, was painted on the skin of mice, and after 30 weeks of exposure (five exposures per week), 80% of the mice had papillomas (Black et al., 1985a). Brown bullheads developed papillomas in a similar experiment with an extract of PAH-rich sediment from the Buffalo River, New York (Black et al., 1985a).

Fabacher et al. (1991) prepared extracts of sediment from four industrialized sites contaminated with PAH, and each extract was separated into two fractions, one containing predominantly nitrogen-containing PAH. Neoplasms developed in medaka exposed to these extracts or fractions from the Black and Fox rivers. The crude extracts caused hepatocellular adenoma and hepatocellular carcinoma, and the nitrogen-containing fraction from the Fox River induced cholangioma and pancreatic ductular adenoma.

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