Contamination is a frequent confounding factor in toxicology studies with anthraquinone and related compounds

Abstract

Anthraquinone (AQ) (9,10-anthracenedione) is an important compound in commerce. Many structurally related AQ derivatives are medicinal natural plant products. Examples include 1-hydroxyanthraquinone (1-OH-AQ) and 2-hydroxyanthraquinone (2-OH-AQ), which are also metabolites of AQ. Some commercial AQ is produced by the oxidation of anthracene (AQ-OX). In the recent past, the anthracene used was distilled from coal tar and different lots of derived AQ often contained polycyclic aromatic hydrocarbon contaminants, particularly 9-nitroanthracene (9-NA). Many toxicology studies on AQ used contaminated anthracene-derived AQ-OX, including a National Toxicology Program (NTP) 2-year cancer bioassay that reported a weak to modest increase in tumors in the kidney and bladder of male and female F344/N rats and in the livers of male and female B6C3F1 mice. The AQ-OX used in that bioassay was mutagenic and contained 9-NA and other contaminants. In contrast, purified AQ is not genotoxic. The purpose of this paper is to provide additional information to help iterpret the NTP cancer bioassay. This paper describes a quantitative analytical study of the NTP anthracene-derived AQ-OX test material, and presents the results of mutagenicity studies with the 1-OH-AQ and 2-OH-AQ metabolites and the primary contaminant 9-NA. Purified 1-OH-AQ and 2-OH-AQ exhibited only weak mutagenic activity in selected strains of tester bacteria and required S9. Literature reports of potent mutagenic activity for 1-OH-AQ and 2-OH-AQ in bacteria minus S9 are, once again, very likely the result of the presence of contaminants in the test samples. Weak activity and limited production of the 1-OH-AQ and 2-OH-AQ metabolites are possible reasons that AQ fails to exhibit activity in numerous genotoxicity assays. 9-NA was mutagenic in tester strains TA98 and TA100 minus S9. This pattern of activity is consistent with that seen with the contaminated AQ-OX used in the NTP bioassay. Analysis of all the mutagenicity and analytical data, however, indicates that the mutagenic contamination in the NTP bioassay probably resides with compounds in addition to 9-NA. 9-NA exhibited potent mutagenic activity in the L5178Y mammalian cell mutagenicity assay in the presence of S9. The positive response was primarily associated with an increase in small colony mutants suggesting a predominance of a clastogenic mechanism. Quantitative mutagenicity and carcinogenicity potency estimates indicate that it is plausible that the contaminants alone in the NTP AQ-OX bioassay could have been responsible for all of the observed carcinogenic activity. Although AQ-OX is no longer commercially used in the United States, many of the reported genotoxicity and carcinogenicity results in the literature for AQ and AQ derivative compounds must be viewed with caution.