Comparative tumorigenicity of the cyclopenta-fused polycyclic aromatic hydrocarbons aceanthrylene, dihydroaceanthrylene and acephenanthrylene in preweanling CD-1 and BLU:Ha mouse bioassays

Jia-Sheng Wang1,2,5, Xia He1,2, Patrick P.J. Mulder3, Ben B. Boere3, Jan Cornelisse3, Johan Lugtenburg3 and William F. Busby Jr2,4

1 Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205, USA,
2 Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA,
3 Leiden Institute of Chemistry, University of Leiden, 2300 RA Leiden, The Netherlands and
4 Gentest Corporation, Woburn, MA 01801, USA


    Abstract
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 Abstract
 Introduction
 References
 
Cyclopenta-fused polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants and potential human health biohazards. In this study, the tumorigenicity of three single cyclopenta-fused polycyclic aromatic hydrocarbons, aceanthrylene, dihydroaceanthrylene and acephenanthrylene, was examined in preweanling CD-1 and BLU:Ha mouse bioassays at total doses of 175, 437.5 and 875 µg/mouse. No death or significant toxicity was observed with the treatment protocol in the tested animals. In CD-1 mice, a significant increase in lung tumor incidence (18–26%, P < 0.025–0.01) for these three compounds was recorded in animals treated with 875 µg as compared with the control animals (3%). Significant numbers of liver tumors (25–41%, P < 0.01–0.001) were induced in all aceanthrylene treatment groups and in animals treated with 875 µg acephenanthrylene (35%) at the termination at 9 months. Most liver tumors were induced in male animals. The ED50 values were estimated as 8.5, 10.6 and 12.8 µmol and the TM1.0 were 15.1, 20.4 and 23.1 µmol for aceanthrylene, acephenanthrylene and dihydroaceanthrylene, respectively. In BLU:Ha mice, there was a significant dose-dependent increase in lung tumor incidence, from 4% for the control group to 33% (P < 0.001) for the animals treated with 875 µg aceanthrylene and to 24% (P < 0.02) for the animals treated with 437.5 µg acephenanthrylene. The ED50 values were 6.0 and 4.4 µmol and the TM1.0 were 9.8 and 6.8 µmol for aceanthrylene and acephenanthrylene, respectively. No significant difference in lung tumor incidence between male and female mice was found. Based on these data and comparisons of tumorigenic potency with other polycyclic aromatic hydrocarbons previously tested in these newborn mouse bioassays, aceanthrylene and acephenanthrylene were classified as weak tumorigens.

Abbreviations: AA, aceanthrylene; AP, acephenanthrylene; BP, benzo[a]pyrene; CP-PAH, cyclopenta-fused polycyclic aromatic hydrocarbon; CPP, cyclopenta[c,d]pyrene; DHAA, 1,2-dihydroaceanthrylene; DMSO, dimethylsulfoxide; ED50, total dose inducing lung tumors in 50% of the treated mice; PAH, polycyclic aromatic hydrocarbon; PMS, post-mitochondrial supernatant; TM1.0, total dose inducing 1.0 lung tumor/mouse in treated mice.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) represent an important class of polycyclic aromatic hydrocarbons (PAHs) that are ubiquitous environmental pollutants (13). Aceanthrylene (AA) and acephenanthrylene (AP) (Figure 1Go) are single cyclopenta-fused derivatives of anthracene and phenanthrene that have been found in many environmental samples. AA has been commonly detected in ambient air (4), water (4) and fuel combustion emissions (5), as well as in indoor air samples from homes in China where coal was burned under unventilated conditions (6). AP has been identified as a product of silica-catalyzed pyrolysis of methane (7) and has often been detected in carbon black extracts, cigarette smoke condensate, tobacco pyrolyzate and fuel combustion effluents (7,8).



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Figure 1. Structures of the test compounds. (I) Aceanthrylene; (II) 1,2-dihydroaceanthrylene; (III) acephenanthrylene.

 
AA was previously reported to be a strong mutagen in the Ames Salmonella reversion assay in tester strains TA98 and TA100 with a rat liver S9 fraction (9). AA has been shown to be a relatively potent mutagen in the Salmonella TM677 forward mutation assay in the absence and presence of post-mitochondrial supernatant (PMS) (8,10). AA was also mutagenic at the thymidine kinase locus (tk) in MCL-3, a metabolically competent line of human B lymphoblastoid cells containing multiple cytochrome P450 enzymes and microsomal epoxide hydrolase (8). AA is metabolized to active intermediates that form relatively stable and persistent DNA adducts in C3H10T1/2 mouse embryo fibroblast cells (11). The major AA–DNA adduct was identified as AA-1,2-oxide–2-deoxyguanosine. However, AA failed to induce detectable morphological transforming activity under the experimental condition of relatively lower doses (11). AP was inactive in the human MCL-3 cell mutation assay and was negative in the Salmonella TM677 forward mutation assay in the absence of PMS (8,10), but it was mutagenic in the presence of PMS in the same assay (8). Although the results of these studies clearly illustrated the mutagenic potential of these compounds in in vitro mutagenic assays, their in vivo tumorigenicity has not been previously demonstrated. In the study reported here, we examined potential tumorigenicity of AA, AP and a derivative of AA (1,2-dihydroaceanthrylene, DHAA) in preweanling CD-1 and BLU:Ha mouse bioassays.

AA and DHAA were synthesized from anthracene (Gold Label, >99.9% pure, CAS no. 120-12-7; Aldrich Chemical Co., Milwaukee, WI) by the method of Olde Boerrigter et al. (12). AP was synthesized from phenanthrene (CAS no. 85-01-8; Aldrich Chemical Co.) by procedures described by Lafleur et al. (8). AA, DHAA and AP were further purified by HPLC and their purities were assessed by gas chromatography–mass spectrometry. High purity glass-distilled dimethylsulfoxide (DMSO) was obtained from Burdick and Jackson Laboratories (Muskegon, MI) and stored under nitrogen.

Pregnant Swiss-Webster BLU:Ha (ICR) mice were obtained from Blue Spruce Farms (Altamont, NY) 5 days before the end of gestation. Time-pregnant (14 days) VAF/Plus CD-1 mice were purchased from Charles River Laboratories (Kingston, NY). The animals were housed individually in polycarbonate tubs equipped with dust filters and containing wood chip bedding in laminar flow isolation cubicles under controlled conditions of temperature (22 ± 1°C), light (12 h light/dark cycle) and humidity (50 ± 10%). NIH open formula diet (NIH-07 Rat and Mouse Feed; Zeigler Bros, Garners, PA) and distilled water were supplied ad libitum. Litters were born 4–6 days after arrival on day 20–22 of pregnancy and were reduced in number to 10 animals each, if necessary. Following the treatment period the pups were weaned at 28 days old and housed according to gender and litter with up to three males or five females/tub under the conditions described above.

Newborn mice were injected i.p. with either AA, DHAA or AP in a total volume of 35 µl DMSO three times over a 2 week period (13). The first injection was on day 1 with 5 µl DMSO containing 1/7 of the total dose, followed by a second injection on day 8 with 2/7 of the dose dissolved in 10 µl DMSO and by the final injection on day 15 with the remaining 4/7 of the dose administered in 20 µl DMSO. Control mice were injected with DMSO using the same protocol. The dose design was based on the solubility of these compounds in DMSO and the maximum soluble concentration (~4.3 µmol) was used as the highest dose level in the assay.

Mice were killed by CO2 asphyxiation at 6 (BLU:Ha) or 9 months of age (CD-1) and necropsied. The skin, musculature and internal organs were examined for visible abnormalities and any tissues with lesions were preserved in buffered formalin. Tumors visible at the surface of the separated lobes of the preserved lungs were counted. Sections of lung tumors were stained with hematoxylin–eosin for histopathological evaluation and classified as either adenomas or adenocarcinomas (13). Tumors in liver or other lesions at different sites were also sectioned, stained and evaluated. Tumor incidence in the lung or liver was expressed as the percentage of animals bearing tumors within a given treatment group. Statistical analysis of tumor incidence was performed by the {chi}2 method as described by Peto et al. (14). The significance of the dose–response trend of lung tumor multiplicity (average no. of lung tumors/animal) was evaluated by Kruskal–Wallis non-parametric analysis of variance.

AA, DHAA and AP were tested for tumorigenicity in CD-1 preweanling mice at total doses of 0.87, 2.17 and 4.33 µmol for AA and AP and 0.86, 2.14 and 4.29 µmol for DHAA, which represent 175, 437.5 and 875 µg/mouse of these compounds. No significant toxicity was observed with the treatment protocol in the tested animals, including the vehicle controls. The percentage of mice surviving between the start of treatment at day 1 after birth to weaning at day 28 was 99% for the vehicle control and treated groups. Survival was nearly 100% in all groups between weaning and termination of the experiments at 9 months. The results from the tumorigenicity study of AA, DHAA and AP in CD-1 mice are presented in Table IGo. No increase in lung tumors was observed in animals treated with low (175 µg) and mid-range (437.5 µg) doses of AA and DHAA, but the high dose (875 µg) induced a significant increase in lung tumor incidence (26% for AA and 18% for DHAA, P < 0.025) and multiplicity (0.31 and 0.20 lung tumors/mouse for AA and DHAA, P < 0.01). Lung tumor incidence was also significantly elevated in AP-treated animals from 0% in the 175 µg group to 21% in the 875 µg treatment group (P < 0.01). There were no statistically significant differences in lung tumor incidence between male and female mice treated with AA, DHAA or AP.


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Table I. Tumorigenicity of aceanthrylene, dihydroaceanthrylene and acephenanthrylene in preweanling CD-1 mice
 
Significant numbers of liver tumors were induced only in all the AA treatment groups and in the high dose AP (875 µg) treatment group (Table IGo). No liver tumors were found in control animals and most liver tumors were confined to treated male mice. Histopathological examination confirmed that all liver tumors in the treatment groups were nodular hyperplasia. No significant lesions in other tissues were recorded.

AA, DHAA and AP were also tested for tumorigenicity in a 6 month BLU:Ha preweanling mouse bioassay. AA was tested at the same three doses as in CD-1 mice. DHAA was tested at only one low dose (175 µg/mouse) and AP was tested at two doses (175 and 437.5 µg/mouse). Again, no apparent toxicity was observed in control and treated animals. Survival was 100% in all groups between day 1 after birth and termination of the experiments at 6 months. As shown in Table IIGo, there was a dose-dependent increase in AA- and AP-induced lung tumors. The lung tumor incidence significantly increased from 4% for the vehicle control group to 33% for the mice treated with 875 µg AA and to 24% for the mice treated with 437.5 µg AP. The number of lung tumors/mouse (tumor multiplicity) increased from 0.04 for the controls to 0.42 for AA and to 0.33 for AP. Histopathological examination confirmed that most of these tumors were benign adenomas, with only a few observations of malignant tumors that were classified as adenocarcinomas. No tumors were found in liver and other organs from both control and treated BLU:Ha mice.


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Table II. Tumorigenicity of aceanthrylene, dihydroaceanthrylene and acephenanthrylene in preweanling BLU:Ha mice
 
Data from the CD-1 and BLU:Ha mouse assays were used to estimate the potency index for lung tumor incidence (ED50) and multiplicity (TM1.0) using a linear extrapolation model of the dose–response curves (15). In CD-1 mice, the estimated ED50 values (total dose inducing lung tumors in 50% of the treated mice) were 8.5, 12.8 and 10.6 µmol and the TM1.0 (total dose inducing 1.0 lung tumor/mouse in treated mice) were 15.1, 23.1 and 20.4 µmol for AA, DHAA and AP, respectively (Table IIIGo). In BLU:Ha mice, the estimated ED50 values for AA and AP were 6.0 and 4.4 µmol and the TM1.0 were 9.8 and 6.8 µmol, respectively.


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Table III. Comparative lung tumorigenic potency of some polynuclear aromatic hydrocarbons in two preweanling mouse bioassays
 
These results (Tables I and IIGoGo) clearly show that AA was tumorigenic in preweanling CD-1 and BLU:Ha mouse bioassays, as demonstrated by the significantly higher incidence of lung and liver tumors in AA-treated animals than in the vehicle control group. The tumorigenicity of AA in mice reported in this study was consistent with previously reported data on its mutagenicity in bacterial and human cell assays (810) and its ability to form DNA adducts in mouse embryo fibroblast cells (11). On the other hand, DHAA, a derivative from saturation of the cyclopenta double bond in AA, was not tumorigenic in the liver of CD-1 mice. The high dose (875 µg) also induced less lung tumors than AA in the same mouse strain (Table IGo). The reduced potency in tumorigenic activity of DHAA was also consistent with recently published data showing significant reduction of mutagenicity for DHAA as compared with AA in a Salmonella forward mutation assay (10).

This study also found that AP caused significant increases in incidence of liver and lung tumors at the high dose (875 µg) tested in CD-1 mice (Table IGo) and that the mid-range dose (437.5 µg) of AP induced a significant number of lung tumors in BLU:Ha mice (Table IIGo). The data were also consistent with AP mutagenic activity in a Salmonella forward mutation assay in the presence of PMS (8), however, its minimum detectable mutagen concentration was about five times higher than that of AA (10). Nevertheless, the tumorigenic activities of AA and AP in this study were similar.

The tumorigenic potency of other compounds in the preweanling mouse bioassay has been evaluated by their TM1.0 and ED50 values in previously published studies (15,17). When compared with other PAHs tested previously in preweanling BLU:Ha and CD-1 mouse bioassays as listed in Table IIIGo, AA and AP in BLU:Ha mice were 20–40 times less potent than benzo[a]pyrene (BP) and 3–6 times less potent than cyclopenta[c,d]pyrene (CPP). AA and AP in CD-1 mice were 126–170 times less potent than BP and 30–40 times less potent than CPP. Based on these comparisons, AA and AP should be classified as weak tumorigens in these mice strains. However, AA and AP in BLU:Ha mice were much more potent than fluoranthene and phenalenone, weak tumorigens and more abundant PAHs in the environment. In CD-1 mice, AA and AP were also ~1.5–2 times more active than fluoranthene.

The work reported here for the evaluation of the tumorigenicity of AA, AP and DHAA was started in the BLU:Ha mouse, which is a sensitive strain for examining the tumorigenic activity of fuel combustion emissions and their components in our laboratory (13,15,17). The same mouse model has also been used by other investigators to test potential proximate and ultimate carcinogenic metabolites of BP and other PAHs (18). Because of the unexpected commercial loss of the BLU:Ha mouse strain, the study was extended to CD-1 mice. We have previously used fluoranthene to validate the CD-1 mouse bioassay in the same dose range and treatment protocol as tested in the BLU:Ha mouse (19). Comparative tumorigenicity data for fluoranthene from two mouse strains revealed that the CD-1 mouse was less sensitive for lung tumor induction than the BLU:Ha mouse. Data obtained from this study (Tables I and IIGoGo) also confirmed that finding, as evidenced by a higher incidence of lung tumors in AA- and AP-treated BLU:Ha mice than in CD-1 mice. In the mid-range dose (437.5 µg) of AA- and AP-treated BLU:Ha mice, the incidence of lung tumor increased ~2-fold above that in CD-1 mice. However, the lung tumorigenic potency of BP and CPP in CD-1 mice was 2–3 times stronger than in BLU:Ha mice by comparison of TM1.0 (Table IIIGo), suggesting that the CD-1 mouse assay may be more sensitive to strong carcinogens and less sensitive to weak carcinogens. In addition, the CD-1 mouse is more sensitive in liver tumor induction, which was not seen in BLU:Ha mice in previous and the present studies.

Strong mutagenicity in bacterial and human cells and potent tumorigenicity in preweanling mouse bioassays are consistent properties of combustion emission extracts and components (8,10,15,1721). Data from this and other studies (8,10) suggest that CP-PAHs like AA and AP may contribute in part to the potent mutagenicity and tumorigenicity found for combustion emission extracts. Because of their widespread environmental occurrence and biological activities, these CP-PAHs could pose potential human health risks, especially AA, which has proved positive in many bioassays so far tested. The metabolic activation of AA and AP in human cells and their combined biological effects with other PAHs in fuel combustion extracts and particulate matter should warrant further investigation.


    Acknowledgments
 
This research was supported in part by grants P01-ES01640 and P01-ES06052 from the National Institute of Environmental Health Sciences. We deeply regret to inform the scientific community of the untimely death of Dr William F.Busby on March 13, 1999. He was a mentor, colleague and friend of many in the toxicology community.


    Notes
 
5 To whom correspondence should be addressed at: Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, 615 North Wolfe Street, Room 1102, Baltimore, MD 21205, USA Email: jswang{at}jhsph.edu Back


    References
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 Abstract
 Introduction
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Received December 3, 1998; revised February 5, 1999; accepted March 1, 1999.





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