Monocyclic Aromatic Amines as Potential Human Carcinogens Old Is New Again
Carcinogenesis. 2010 Jan; 31(1): fifty–58.
Monocyclic aromatic amines as potential man carcinogens: one-time is new once more
Received 2009 Sep 22; Revised 2009 Oct 24; Accepted 2009 Oct 26.
Abstruse
Alkylanilines are a grouping of chemicals whose ubiquitous presence in the surroundings is a result of the multitude of sources from which they originate. Exposure assessments indicate that most individuals experience lifelong exposure to these compounds. Many alkylanilines accept biological activity similar to that of the carcinogenic multi-ring aromatic amines. This review provides an overview of human exposure and biological furnishings. It also describes contempo investigations into the biochemical mechanisms of action that lead to the cess that they are almost probably more complex than those of the more than extensively investigated multi-ring aromatic amines. Non merely is nitrenium ion chemistry implicated in Deoxyribonucleic acid damage by alkylanilines but also reactions involving quinone imines and perhaps reactive oxygen species. Recent results described here point that alkylanilines can be potent genotoxins for cultured mammalian cells when activated by exogenous or endogenous stage I and phase II xenobiotic-metabolizing enzymes. The nature of specific DNA damage products responsible for mutagenicity remains to exist identified but evidence to date supports mechanisms of activation through obligatory N-hydroxylation as well equally subsequent conjugation by sulfation and/or acetylation. A fuller understanding of the mechanisms of alkylaniline genotoxicity is expected to provide of import insights into the environmental and genetic origins of i or more than human cancers and may reveal a substantial role for this group of compounds every bit potential human chemical carcinogens.
Introduction
Aromatic amines are a class of organic compounds that include many members that are carcinogenic, both experimentally in the research setting and experientially in human life. The carcinogenesis literature documents innumerable studies demonstrating that administration of a considerable variety of aromatic amines to experimental animals of different species induces cancers in those animals (one). The epidemiological literature leaves little doubt that a specific few aromatic amines are the crusade of bladder cancer in occupationally exposed persons and there is a convincing argument to be made that exposure to effluvious amines via tobacco fume is a major, if not predominant, gene in causing float cancer in smokers (2).
The biochemical mechanisms by which effluvious amines might induce cancers have been investigated extensively and are now thought to be reasonably well understood (3–5). Homo population studies that take incorporated measures of metabolic genotype and phenotype tend to back up the biochemical mechanisms inferred from experimental studies (6). It appears that, for this class of chemical carcinogens, the linkage betwixt the experimental setting and the homo condition is as strong as whatsoever. Notwithstanding, there remains a conundrum: outside the occupational setting there appears to exist far too little exposure to any of the known human being float carcinogens to business relationship for the observed incidence rates.
Various possibilities may be considered for resolving this conundrum. The models used for extrapolating from high to low dose may underestimate dominance at homo exposure levels. There may be ecology exposures to known compounds that are largely cryptic or in that location may be amines in the environment that have not yet been identified. Unanticipated synergies among multiple exposures may occur. Other possibilities no doubts exist. Because of their environmental prevalence, one relatively unexplored group of aromatic amines, the alkylanilines, may exist significantly involved in one or more of these explanations. In this paper, we will review much of the research on alkylanilines that addresses the question of why they could exist playing an important office in human cancer.
Environmental prevalence
In considering the importance of any specific chemical carcinogen or grade of carcinogens to human disease incidence, weight must be given to the extent to which the compounds are distributed in the surround. Alkylanilines, as a course, announced to exist distributed widely, making exposure to them virtually ubiquitous. Early efforts at exposure cess were predicated on the fact that alkylanilines are present in tobacco smoke (vii–ix). Accordingly, indoor spaces contaminated with tobacco smoke exhibited higher levels of alkylanilines than uncontaminated spaces (8,10). Outdoor air was generally lower with exceptions that may be attributable to industrial sources (10). Exposure assessment based on hemoglobin adduct levels also indicates that tobacco fume is a significant, if not predominant, source for many alkylanilines (xi,12). In both studies, 2,6-dimethylaniline (two,6-DMA) was an exception, being really higher in not-smokers than smokers in a study conducted in Italia (eleven). Much higher levels (7- to 8-fold) of 3,5-dimethylaniline (three,5-DMA) were besides observed in not-smokers in the Italian population. This finding attains greater significance in low-cal of a recent investigation in Canada (13) that institute levels of 3,5-DMA in indoor and outdoor air that are two to iii orders of magnitude higher than those reported elsewhere.
Exposure assessment through hemoglobin adduct analysis has now been extended to include non-tobacco sources in the U.s. (12), Italy (11), Deutschland (fourteen) and about recently by united states of america in China (unpublished results). In all of these studies, there have been virtually no subjects that do not have hemoglobin adducts of all of the compounds investigated. Results tend to be comparable: while there may exist a considerable range of values among individual subjects, mean values tend to fall within a range of <10-fold. The inescapable determination from these results is that environmental prevalence of many alkylanilines is extensive and that exposure to them is not confined to limited population subgroups such as the occupationally exposed.
Figure 1 illustrates most of the of import routes by which alkylanilines come to be present in the environment. It is based on the comprehensive information about production and possible releases into the surround of specific alkylanilines that can be found in the Hazardous Substances Data Banking concern (http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB) every bit well every bit the references cited higher up.
Ecology sources of alkylanilines.
Evidence for carcinogenicity in a population-based written report
In 2004, we published results from a population-based written report of bladder cancer conducted in Los Angeles county in which chemical markers of exposure to a specific set of alkylanilines were included (12). This was the first, and is yet the only, population-based bladder cancer written report of any effluvious amines having the basic aniline ring structure. In the absenteeism of additional studies with similar findings, the results are not definitive, but considering the power of the report, we were strongly motivated to pursue boosted mechanistic and in vitro toxicology studies in an endeavor to provide biological plausibility for the epidemiological findings.
The Los Angeles study examined exposure to nine unlike alkylanilines, including all three ethyl and all half dozen dimethyl isomers. Statistical assay establish that three of the ix—2,6-DMA, 3,5-DMA and 3-ethylaniline (iii-EA)—were independently associated with chance for development of float cancer. Others were also associated with run a risk but not independently as there were strong correlations among the various anilines as well as with four-aminobiphenyl exposure. Chiefly, no meaningful differences were found when analysis was restricted to lifelong non-smokers, implying that smoking was not a confounder in this study.
Evidence for carcinogenicity from occupational exposure
In that location exist only very limited information from occupational studies that comport direct on the human carcinogenicity of monocyclic amines and none specifically related to the alkylanilines discussed here. The closest analog that has been evaluated comprehensively is o-toluidine. In a report of workers exposed to aniline and o-toluidine, it was found that levels of exposure to these chemicals were associated with a statistically significant increment in incidence of bladder cancer (15). The initial study did not distinguish between aniline and o-toluidine exposure but argued that the latter was more important. The written report was challenged (16,17) on the grounds that prior exposure of the same workers to more potent chemicals could non be ruled out or could ongoing exposure to 4-aminobiphenyl (4-ABP). The effect of prior exposure was rebutted (xviii,xix) and the issue of ongoing 4-ABP exposure was addressed through a follow-upward study in which current exposure to aniline, o-toluidine and 4-ABP was assessed through biomonitoring of hemoglobin adducts (xx). Biomonitoring clearly revealed that iv-ABP exposure was independent of workplace exposure condition and, as observed in numerous other studies, was more often than not associated with cigarette consumption. Thus, the most likely finding of this retrospective accomplice study is that o-toluidine is indeed a human float carcinogen. A follow-up study at the same chemical institute reported in 2004 (21), long afterwards potential co-exposures to other chemicals is idea to accept ceased, revealed a continuing excess of bladder cancer among exposed workers.
Experimental carcinogenicity
A comprehensive review of the carcinogenicity of alkylanilines is outside the scope of this newspaper mainly because so many of the results, while suggestive of carcinogenic potential, are largely inconclusive. Furthermore, the National Toxicology Program has recently conducted a review of the literature as part of its process for nominating several alkylanilines for hereafter study. The review can exist found in the nominating document at http://ntp.niehs.nih.gov/ntp/noms/Support_Docs/Alkylanilines060809.pdf. The document includes genotoxicity data every bit well and therefore covers an expanded set up of fourteen compounds. Here, we will focus on the ii compounds for which the International Agency for Cancer has institute sufficient evidence to categorize them every bit either carcinogenic to humans (o-toluidine; Group ane) or possibly carcinogenic to humans (2,half dozen-DMA; Group 2B).
As described by the International Agency for Cancer, 'ortho-toluidine was tested for carcinogenicity as its hydrochloride salt in two experiments in mice and in three experiments in rats and as the gratis base of operations in i limited experiment in hamsters. Later oral administration to mice, it induced an increased incidence of haemangiomas and haemangiosarcomas and hepatocellular carcinomas or adenomas. In rats, oral administration of ortho-toluidine increased the incidence of tumours in multiple organs, including fibromas, sarcomas, mesotheliomas, mammary fibroadenomas and transitional cell carcinomas of the urinary bladder'. Based on these results, the International Bureau for Cancer has concluded that there is sufficient show for the carcinogenicity of o-toluidine in experimental animals. The lack of organ specificity casts some incertitude on the significance of this conclusion but the occurrence of transitional cell carcinomas of the urinary bladder is notable, as this is typical of many effluvious amine carcinogens.
In contrast to o-toluidine, two,6-DMA is an organ-specific carcinogen, inducing carcinomas and papillary adenomas of the nasal crenel in male person and female rats in a dose-dependent manner (22). Subsequent studies strengthen the bioassay findings. When [fourteenC]-2,half dozen-DMA was given to rats, Deoxyribonucleic acid in the ethmoid turbinate as well as in liver became radiolabeled (23). In contrast to hepatic DNA, labeling of the ethmoid turbinate DNA was only observable later on pretreatment with unlabeled 2,6-DMA, indicating that the amine induced an enzyme present in the nasal cavity that is responsible for its activation and that the same enzyme is a minor constituent in liver. Contempo studies in our laboratories support this inference: when various sources of P450s were screened for their ability to convert 2,vi-DMA to the corresponding hydroxylamine, rat liver microsomes exhibited undetectable action, whereas human liver microsomal activity was readily credible (24; unpublished results). Of five recombinant human being P450s tested (1A1, 1A2, 1B1, 2A6 and 2E1), simply 2A6 exhibited N-hydroxylation activeness. Notably, one fellow member of the 2A family, 2A3, appears to exist expressed preferentially in the rat nasal cavity (25).
Chemic reactivity: comparing of alkylanilines with multi-ring carcinogenic amines
A paradigmatic model for aromatic amine genotoxicity has evolved over the last forty years from the seminal work of James and Elizabeth Miller and their coworkers that is described in detail in several afterwards publications (v,26,27). In this model, aromatic amines (and amides) first undergo P450-catalyzed oxidation at the nitrogen atom. The resulting Due north-hydroxylamine has the potential to undergo N–O bond heterolysis to produce a reactive nitrenium ion that reacts with nucleobases in DNA to induce genotoxicity. Heterolysis is promoted in most cases by the intermediate step of phase Ii conjugation of the hydroxyl group just may also be catalyzed by protonation.
In view of the apparent genotoxicity or carcinogenicity of at least some aniline derivatives, several groups have undertaken investigations to determine whether the nitrenium ion mechanism of Dna damage is operative for unmarried-ring effluvious amines. Certainly, DNA adducts of the same basic structure as formed by multi-ring amines can be synthesized via nitrenium ions. Aniline, the simplest effluvious amine, forms the dG–C8 adduct when it is present in the reaction in the activated form, Due north-acetoxyaniline (28). Marques et al. get-go reported the synthesis of dG–C8 adducts of o-, grand- and p-toluidine, ii,3-dimethylaniline and two,4-dimethylaniline by reaction of the corresponding N-(acyloxy)arylamines with dG, dG nucleotides and DNA, where the acyl group was either acetyl or pivaloyl (29). They later reported dG adducts of all the isomeric methyl, ethyl and dimethylanilines using the same synthetic methods (xxx). dG adducts of p-chloroaniline and p-anisidine have also been described equally products of synthesis via nitrenium ion chemistry (31). Jones et al. (32) later on expanded the range of substituted anilines that class dG adducts through their acyloxy derivatives to include ii-chloroaniline. The structures of these and the other adducts described below are shown in Figure 2.
Structures of the various alkylaniline DNA adducts that take been elucidated.
A more detailed study of 2,6-DMA revealed that not just does this aniline form C8 adducts with dG but as well its acyloxy derivative reacts with DNA to form two other dG adducts (33). One occurs by forming a bond between the para-carbon and O half dozen of the nucleobase, whereas the other occurs past attack at N 2. 1 dA adduct is also formed, again by set on of the para-carbon, in this case at North half dozen of the nucleobase. In our own work, we found that iii,5-DMA, when converted to the acetoxy derivative, produces four distinct adducts in Dna (34). 1 was the expected dG adduct. The analagous dA–C8 adduct as well occurred. In addition, there was the adduct formed by attack of the para-carbon at North half dozen of adenine, as was reported for ii,vi-DMA. Lastly, nosotros found an adduct formed with dC through attack of the nitrogen cantlet at C5 of the nucleobase. Despite the novelty of ii of the adducts, all were consistent with the intermediacy of a nitrenium ion.
Demonstration that the Deoxyribonucleic acid adducts expected from nitrenium ion chemistry do occur when DNA is treated with appropriately activated alkylanilines may be considered a necessary condition to expect their occurrence in vivo following exposure to the parent amines. Following up on these demonstrations, several attempts have been made to detect the chemically synthesized adducts in treated animals. Jones et al. (32) developed a method for quantitative analysis of dG and dA adducts of a set of aromatic amines that included the three toluidines, 2,4-dimethylaniline and 2,six-DMA. The method was based on HPLC/MS/MS and could distinguish among different adduct structures. When hepatic DNA from rats given 0.five mmol/kg of the compounds was analyzed, no adducts of any of the monocyclic amines were detected above the level of 3.ii adducts per 108 normal bases. A polycyclic aromatic amine, 4-aminobiphenyl, was included for comparison and it produced adducts at ∼500 per ten8 bases.
Adopting a unlike strategy (35), nosotros treated wild-blazon C57/BL6 mice with [14C]-labeled 2,half-dozen-DMA, three,5-DMA and 3-EA and analyzed tissue DNA for isotopic labeling by accelerator mass spectrometry. All the compounds produced detectable labeling of Deoxyribonucleic acid at levels that ranged up to more than ane per 10vii bases for 3,v-DMA in liver. In this experiment, the dose of alkylaniline was ∼ane μmol/kg, which is 1/500th of the dose given in the experiment described above where no adducts were detectable. Only one alkylaniline, two,vi-DMA, is common to both studies. In our study, it besides produced adducts at the level of 1 per ten7 bases but only at 4 h mail-dosing; at 24 h—the time betoken used in the other report—the level had declined to about i per 108 bases. While this level is below the limit of detection past the HPLC/MS/MS method, the divergence in dose should be kept in listen when comparison the two studies.
Since the analytical method we used, accelerator mass spectrometry, was capable only of detecting isotope, it cannot be inferred that any of the adducts detected were structurally identical with whatever of the adducts produced past nitrenium ion reactions. To gain further insight into the nature of the bound isotope, we take been performing high-performance liquid chromatography analyses of enzymatic digests of the Deoxyribonucleic acid isolated from tissues harvested from animals given [14C]-labeled iii,5-DMA. The choice of 3,5-DMA-treated animals was governed by the availability of nitrenium adducts of defined structure as described above (34). Digests of hepatic Deoxyribonucleic acid were combined with a mixture of the four adduct standards at a level that produced well-defined UV-detectable peaks in the chromatogram and subjected to high-performance liquid chromatography analysis with fraction drove. Accelerator mass spectrometry analysis of the collected fractions was performed to generate a radiochromatogram that could be compared with the UV trace. Several such analyses have been conducted with inconsistent results in the radiochromatograms. In none of the analyses, though, have we observed co-chromatography of radiocarbon with UV peaks produced past the standards, suggesting that if any of the DNA adducts formed in vivo are identical to the standards they are present just as pocket-size constituents.
Thus, far, picayune evidence has been developed to back up a model for genotoxicity of alkylanilines that is comparable with the very successful model that characterizes multicyclic aromatic amine carcinogenicity. Nonetheless, the bachelor data is far from sufficient to dominion the model out and the ability of nitrenium ions derived from monocyclic amines to form Dna adducts is reason to remain open minded. In the adjacent department, then, we will prove that North-hydroxylation, a critical metabolic footstep for activating many aromatic amines, is important also in monocyclic amine genotoxicity.
N-hydroxylation as a critical activation step
At that place is aplenty show that alkylanilines and other monocyclic amines are readily N-hydroxylated by mammalian metabolism. In peradventure, the virtually extensive investigation of the phenomenon to date, Sabbioni (36) tested 32 compounds in rats for their ability to bind to hemoglobin in a mode that is demonstrative of the generation of N-hydroxylamines in vivo. Just one of the 32 appeared to exist resistant to metabolic transformation to a hemoglobin-binding hydroxylamine. Data from human studies are similarly indicative that alkylanilines are nearly-universally susceptible to metabolic N-hydroxylation (11). Direct evidence for production of an N-hydroxylamine in vivo in rats has also been presented by Kulkarni et al. (37), who demonstrated urinary excretion of N-hydroxy-o-toluidine post-obit assistants of the amine.
Information technology has thus widely been assumed that generation of N-hydroxylamines in vivo is associated with genotoxicity but never demonstrated that N-hydroxylation is a critical step in leading to DNA adduct formation or harm. We recently undertook a multi-species multi-compound report to assess the role of Northward-hydroxylation (38). Using S9 from human, rat, dog and mouse liver, we determined the dependence of DNA adduct formation by three alkylanilines in vitro on the presence of NADPH. Results and experimental details are given in Table I. It is articulate from the data that NADPH is a nearly absolute requirement for Deoxyribonucleic acid adduct formation to occur. Whether the levels observed in the absenteeism of NADPH represent true adduct formation is hard to assess. Command experiments were conducted in which [14C]alkylanilines were non added until immediately before terminating the incubation in lodge to appraise not-covalent binding. Results from those experiments were comparable with results from experiments in which NADPH was omitted; thus, information technology is not credible that any adduct formation occurred in the absence of NADPH.
Table I.
Formation of DNA adducts by three [xivC]alkylanilinesa
| 3-EA | 3,5-DMA | ii,vi-DMA | ||
| Human | Control | fifteen ± half dozen | 36 ± 11 | thirteen ± 1 |
| Experiment | 15 ± 3 | 1392 ± 74 | 258 ± 187 | |
| Rat | Control | 39 ± 12 | 5 ± 1 | 11 ± 1 |
| Experiment | 30 ± 5 | 1101 ± 204 | fifty ± 39 | |
| Dog | Control | 15 ± viii | 21 ± 14 | 35 ± 21 |
| Experiment | 22 ± 1 | 727 ± 37 | 180 ± 53 | |
| Mouse | Control | 68 ± 16 | 77 ± 23 | 33 ± 2 |
| Experiment | 162 ± 9 | 139 ± 35 | 146 ± 22 |
Not all the ramifications implicit in the data given in Table I take been explored in item just one feature in detail deserves further word. Footling activation of 3-EA to DNA-binding metabolites was observed using S9 from three of the four species, mouse being the exception. This finding was discordant with results for N-hydroxylation, which indicated that iii-EA was mostly N-hydroxylated at a charge per unit several times greater than the rates observed with 3,v-DMA or ii,6-DMA (38). To explore this discrepancy farther, Deoxyribonucleic acid bounden was measured at lower concentrations of alkylanilines using human S9. At 25–1000 nM, the same order of reactivity was observed as at 25 μM simply the differences between 3-EA and the other ii alkylanilines were much smaller, ranging from nearly identical to ∼6-fold less (Figure 3). North-hydroxylation rates at the lower substrate concentrations have not been measured; and then we tin can only speculate about the reasons for the alter in relative DNA adduct formation. Ane possibility is that generation of the ultimate electrophilic species from iii-EA, whether directly from the N-hydroxylamine or by a unlike pathway, becomes kinetically favored at lower substrate concentration. Whatsoever the actual explanation, the results are equivocal with regard to the part of N-hydroxylamines in Dna adduct formation.
Formation of Deoxyribonucleic acid adducts catalyzed past human S9 at low doses. Experimental details given in footnote to Table I except that the alkylaniline concentrations used were 25, 100 and grand nM.
Further studies to endeavour to demonstrate interest of N-hydroxylamine derivatives in Dna harm involved manipulating in vitro activation systems with respect to phase 2 metabolism. Using the same three alkylanilines listed in Table I, we assessed DNA bounden of radioisotope catalyzed past man liver S9 in the absence and presence of cofactors that promote N-acetyltransferase (NAT), sulfotransferase and glucuronyl transferase activities. Results are presented in Figure 4, along with experimental details.
Effect of Phase Ii conjugation activity on DNA adduct formation in vitro. NAT: All incubations were conducted in 0.2 ml 50 mM Tris buffer (pH seven.four) containing 1 mg/ml ct-Deoxyribonucleic acid, ii mg/ml homo liver S9 fraction, NADPH-regenerating system and 1 μM of the respective [14C]amine for 6 h at 37°C. Supplementation was as follows: NAT, one mM ethylenediaminetetraacetic acid, i mM dithiothreitol, 1.08 mg/ml acetyl-D,L-carnitine, 0.22 U/ml carnitine acetyltransferase and 0.ane mM acetyl coenzyme A; SULF, 0.one mM PAPS and UGT, 8 mM MgClii, 25 μg/ml alamethicin and 2 mM UDPGA.
Promotion of NAT activity did not have the aforementioned effect on all the compounds tested. Information technology produced a hit negative event on bounden by ii,vi-DMA, decreasing the adduct level past 78%. The issue on 3-EA binding was besides negative but considerably smaller at 28%. In contrast, the effect with 3,five-DMA was to raise adduct germination by 53%. Decreased binding nether the conditions of the experiment is uninformative with respect to role of an N-hydroxylamine in Dna adduct formation because the non-oxidized amine is frequently a substrate for NAT. Enhancement, though, is a better indicator because O-acetylation of the hydroxylamine promotes its reactivity. Results appear to indicate a significant role for the N-hydroxylamine of 3,5-DMA in Deoxyribonucleic acid bounden (just, meet comments on Bamberger rearrangement beneath).
Likewise, the promotion of DNA binding of all iii alkylanilines by sulfotransferase activity points to a pregnant role for N-hydroxylation since it would be expected to produce sulfate esters, which, like acyl esters, are more reactive than the unconjugated hydroxylamines. Decreased bounden every bit a event of promotion of glucuronyl transferase activity is largely uninformative, again because the not-oxidized amine can exist a substrate.
Other mechanisms—N-hydroxylation every bit a beginning footstep to other genotoxins
Largely overlooked until recently is the potential beingness of other chemic reaction pathways leading to Deoxyribonucleic acid adducts or other Deoxyribonucleic acid impairment that do not directly involve electrophilic nitrenium ions. Phenols are prominent products of aromatic amine metabolism that may arise past straight oxidation of the aromatic band or, equally others accept conjectured on theoretical grounds (39), from the radical cation that is the forerunner to the N-hydroxylamine as shown in Figure 5. The phenols have mostly been regarded as detoxification products. To the extent that the phenols are afterwards conjugated with sulfate, this is probably accurate because sulfates are highly polar h2o-soluble compounds. Moreover, with the acidic proton of the phenol no longer nowadays, the aminophenol is highly resistant to oxidation. If the phenolic oxygen is not conjugated, the aminophenol tin can undergo spontaneous or peroxidase-catalyzed oxidation, as shown in Figure five, to a quinone imine, which is highly reactive as an electrophile and can undergo redox cycling equally well.
Generation of aminophenols/quinone imines from N-hydroxylamines. The transformations shown hither are alternative routes to aminophenols produced by directly band oxidation by P450s.
Significantly, complicating any analysis of the office played past aminophenol products of metabolism is the fact that these compounds tin can besides course by rearrangement of existing N-hydroxylamines. As a chemical reaction, this transformation is referred to equally the Bamberger rearrangement and requires strong acid catalysis. It also proceeds in biological systems, where the rearrangement tin can be catalyzed past cytochrome P450 2E1, alcohol dehydrogenase and perhaps other enzymes as discussed or referenced in ref. (40). Thus, whenever an N-hydroxylamine is introduced into a biological system, the possibility that information technology is converted into an aminophenol before it undergoes any direct reaction with a biological target must be considered.
Straight evidence of genotoxicity of quinone imines has been reported (41) from studies that measured sister chromatid exchanges in cultured human being lymphocytes. Of particular notation is that Hill et al. included the p-quinone imine derived from 2,half dozen-DMA in the report and constitute that it induced sis chromatid exchanges at doses as low equally 0.i μM. Virtually of the evidence, though, for biological activeness of quinone imines or aminophenols comes from studies of other toxic effects.
Coexisting prove for a possible role of quinone imines in alkylaniline cytotoxicity comes from studies of acetaminophen linking the transcription factors Nrf2 and Keap1 and toxicity. Oxidation of acetaminophen to N-acetyl-p-benzoquinone imine is idea to be largely responsible for the hepatoxicity of this widely used analgesic. Nrf2-nix mutant mice consequently exhibit increased sensitivity to acetaminophen hepatoxicity (42,43). More recently, it has as well been demonstrated that Keap1-knockout mice acquire resistance against acetaminophen hepatoxicity through activation of Nrf2-antioxidant-responsive element pathways (44). Similarly, information technology has likewise been shown that acetaminophen can initiate nuclear translocation of Nrf2 in vivo in mice (45) and that it is then functionally active.
Aminophenols are amend known as nephrotoxins than genotoxins but their mechanistic actions may exist like in that they are electrophilic reactants. The chemical reaction pathways shown in Effigy vi are presented every bit an illustration of current hypotheses to explicate the nephrotoxicity and nephrocarcinogenicity of aminophenol/quinone imines as well every bit the closely related hydroquinone/quinones. At present, available information support a machinery in which oxidation and glutathione conjugation occur in the liver. Mono-, bis- and tris-glutathionyl adducts are exported and taken upwardly by the kidney where conversion to mercapturic acids occurs to a variable extent. Lastly, the thioether conjugates become leap to proteins through the aforementioned Michael reaction that produces the glutathione adducts or through add-on–emptying reactions in which the poly peptide displaces one of the thioether moieties. Examples of each of these steps have been reported (46–48), including the formation of multiple glutathione adducts of aminophenol (49) as well as the aminophenol metabolite of 3-(North-phenylamino)propane-one,2-diol considered responsible for toxic oil syndrome (50). Farther studies (51,52) have probed the molecular biological furnishings of 2,3,five-tris-(glutathion-South-yl)hydroquinone (TGHQ) also as effects on jail cell signaling and mutagenesis (53–55). Other important findings discussed in Shao et al. (56) indicate that p-aminophenol nephrotoxicity in vivo is mediated by extrarenal bioactivation comprised of both oxidation and glutathione conjugation. A total mechanistic understanding of the toxicity of aminophenols has not yet been adult, but the evidence at this stage points to a central role for quinone imines. Whether they besides can react straight with DNA to produce genotoxic outcomes remains an open question.
Germination of protein adducts past aminophenols. A primal question posed past this article is whether similar reactions occur betwixt aminophenols and DNA.
Studies on the furnishings of TGHQ on cell signaling are of additional interest because they suggest however another machinery through which these species may be capable of producing toxic responses: generation of reactive oxygen species (ROS). TGHQ was reported to induce mutation spectra in the supF factor replicated in human AD293 cells and bacterial MBL50 cells that were similar to those induced by hydroxyl radical (53), ane of several ROS. TGHQ reportedly modulates 12-O-tetradecanoyl phorbol 13-acetate responsive element and nuclear factor-kappaB-binding activity in an ROS-dependent style (54) and exhibits other ROS-inducible events such as posttranslational modification of Bcl-2 and subcellular localization of Bax, culminating in the release of cytochrome c and caspase activation (55). Whether similar effects could be elicited by glutathione conjugates of quinone imines remain unexplored simply considering the very close similarity betwixt quinone imines and quinones it seems probably that generation of ROS by the former occurs in vivo.
Bear witness of alkylaniline genotoxicity: recent in vitro studies in mammalian cells
Genotoxicity of alkylanilines has received very limited investigation to date, and the fragmented available database supports only express conclusions. Combined evidence produced in cultured hepatocytes and prison cell-free systems, some of which has been discussed higher up, prove that both stage 1 (N-hydroxylation) and stage two metabolism (mainly sulfation or acetylation) are required for activation of the alkylanilines to DNA-binding forms. Proposed metabolic pathways leading to the formation of genotoxic DNA harm products of two,half-dozen-DMA are summarized in Effigy 7. The proposed pathways provide the rationale underlying our experimental approaches to characterize mechanisms of mutagenesis and cytotoxicity of this grade of chemicals.
Proposed metabolic pathways leading to germination of genotoxic DNA damage products of 2,6-DMA. Modified from Gan et al. (24).
Most previous investigations accept focused on 2,half dozen-DMA, in view of its demonstrable carcinogenic activity for the nasal cavity of rats. Data published to engagement can exist briefly summarized as follows. In the Salmonella typhimurium assay, 2,3-, 2,iv-, 2,v- and 3,iv-dimethylanilines were weakly mutagenic and caused Deoxyribonucleic acid damage in mammalian V79 cells (57). In the same assay procedure, two,6-DMA was neither mutagenic in multiple bacterial strains in the presence or absenteeism of exogenous activating systems nor was it active in a DNA harm and repair assay in Escherichia coli. It induced mutations in mouse L5178Y lymphoma cells and sister chromatid exchange and chromosomal aberrations in Chinese hamster ovary (CHO) cells with and without exogenous metabolic activation (58,59). No genotoxic upshot in East.coli K12 was observed in a host-mediated assay after feeding of 2,half-dozen-DMA to mice, nor was feeding or injecting it effective in a sexual activity-linked recessive lethal assay in Drosophila melanogaster (58). No increment of micronuclei was detected in the bone marrow of male mice after oral doses of upward to 375 mg/kg/day (60) and it did not induce unscheduled Deoxyribonucleic acid synthesis in rat hepatocytes (61).
North-hydroxy-two,6-DMA, the proposed product of metabolic activation of two,6-DMA in vivo, was mutagenic in S.typhimurium TA100 (62) and was the most mutagenic fellow member of a serial of N-hydroxylated aniline derivatives in the absence of metabolic activation (xxx,63). N-hydroxy-3,5-DMA was mutagenic in the HPRT gene of human lymphoblastoid TK6 cells (run across beneath; 64).
Considering of the diversity of exposure conditions, target genomes and host cells in which mutagenic events produced by carcinogenic aromatic amines take been studied, extrapolation of observations from one organization to another entails substantial doubtfulness. The spectra of Dna lesions created likewise differ qualitatively and quantitatively, and the modified bases identified in any given experiment probably represent only a subset of lesions actually present. Thus, information technology is hard to formulate valid generalizations regarding mutagenic efficiency of specific Dna lesions or mechanisms through which they are created. In view of the widespread human exposure to alkylanilines and other show summarized in the preceding sections, we have undertaken investigations to expand the database apropos their genotoxicity in mammalian cell systems. Nosotros are systematically investigating mutagenic responses and mutation spectra induced by metabolites of alkylanilines that have received little previous study. Evaluations are being washed in well-defined genetic targets and mutagenesis models exposed under controlled weather condition with the ultimate objective of identifying Dna alterations responsible for specific mutational events. To date, our studies of cytotoxicity have focused exclusively on cell survival, assessed by dye exclusion 24 h later on exposure to test compounds.
We have focused on three members of this group, namely 3-EA, 2,6-DMA and iii,five-DMA, and their N-hydroxy and aminophenol metabolites synthesized as described above. Initial studies conducted in homo lymphoblastoid TK6 cells revealed that three-EA and N-hydoxy-3-EA did not cause reduced viability and were not mutagenic to cells exposed to millimolar concentrations and they take not been studied further. In dissimilarity, ii,6- and iii,5-DMA both induced cell expiry, but exhibited considerable variation in authorisation, in which the aminophenol metabolites were considerably more potent than the respective Northward-hydroxylamines. Also, each metabolite of 3,five-DMA was more toxic than its ii,6-DMA counterpart. When these compounds were tested for HPRT mutagenicity, both the N-hydroxy and aminophenol of 3,5-DMA were conspicuously mutagenic but these derivatives of two,six-DMA were not agile over the same range of dose levels (64).
We have also studied cytotoxicity and mutagenesis induced by the alkylanilines and their major metabolites in AS52 cells, which were derived from HPRT− CHO cells, and contain a functional single re-create of the East.coli gpt gene in an SV40-pBR322 plasmid stably integrated into their genomic Dna (65–68). The gpt cistron encodes the purine salvage pathway enzyme xanthine-guanine phophoribosyl transferase, analagous to mammalian HPRT, and thus provides the basis for 6-thioguanine selection of mutants. The arrangement is highly suited for determining mutational spectra induced by chemical mutagens of different types. Results to engagement show that 2,6-DMA and three,5-DMA when activated by a homo liver S9 preparation were both cytotoxic and mutagenic, showing linear dose-response relationships in both parameters at exposure levels between x μM and 1 mM. Their N-hydroxy derivatives were also cytotoxic and mutagenic, only had higher potency, showing comparable dose-related responses at exposure levels between 10 and 100 μM. Similarly, their aminophenols were both cytotoxic and mutagenic and were substantially more potent, causing most 100% jail cell killing and significant mutagenicity at levels below 10 μM. Mutation spectra induced past these treatments are currently under investigation.
We are too characterizing alkylaniline genotoxicity in cell systems designed to provide further information about their metabolic activation. Felton et al. (69,seventy) developed CHO-derived prison cell lines in which both phase I and phase Two enzymes were introduced and have shown these cells to be highly sensitive to mutagenesis by the heterocyclic amine ii-amino-1-methyl-6-phenylimidazo[4,v-b]pyridine. CHO AA8 cells served equally the original source of the UV5 line, which lacks nucleotide excision repair (NER) due to an induced defect in the XPD (ERCC2) cistron (70,71). Transfection of UV5 cells with mouse P450 1A2 complementary Deoxyribonucleic acid established the UV5P3 line (72), which was subsequently transfected with human aryl sulfotransferase (HAST1) or NAT2 from a human being liver complementary DNA library (seventy,73), producing CHO cells that are NER deficient (or proficient) and express mouse P4501A2 plus human aryl sulfotransferase or NAT genes. Based on their heterozygosity at the aprt locus, they can be used for mutational analysis based on selection with 8-azaadenine.
Cells were treated with the parent alkylanilines ii,6-DMA and 3,5-DMA at exposure levels of fifty μM–1 mM or with N-hydroxy or aminophenol metabolites at 5–500 μM. Cell viability was determined 24 h after treatment by trypan bluish exclusion and mutant fraction by 8-azaadenine selection. Major findings of studies to date can exist summarized as follows. The influence of NER condition was investigated by comparison of responses of AA8 CHO cells, which are NER proficient, and UV5 cells, which are NER deficient. Neither jail cell line expresses significant levels of phase I nor phase II enzymes. In the absence of human liver S9, the parent alkylanilines were cytotoxic, but not mutagenic, at exposure levels of 100 μM–one mM, as expected. Interestingly, cytotoxicity was enhanced by NER deficiency, suggesting that Dna damage by products that were not mutagenic in the assay used contributed to the toxicity of the compounds. The Northward-hydroxy derivatives of 2,6-DMA and 3,v-DMA were likewise toxic, causing dose-related loss of viability at doses between 100 μM and 1 mM; toxic potency was substantially increased by NER deficiency, resulting in like loss of viability at levels of 10–200 μM. These derivatives were not mutagenic at doses upwards to 1 mM. Responses to the aminophenol derivatives were virtually identical to those of the Due north-hydroxylated forms, i.due east. they were potent inducers of cytotoxicity, which was enhanced past NER deficiency, but not mutagenic under these conditions.
The influence of NER status was also studied by several other comparisons. In two cell lines both expressing CYP1A2 and NAT2, but 1 scarce in NER, 2,half dozen-DMA and 3,5-DMA were cytotoxic and mutagenic at dose levels between 100 μM and 1 mM. Their potency in inducing both responses was greatly enhanced in the NER-deficient cells. In two cell lines both deficient in NER and ane expressing CYP1A2 and NAT2, 2,6-DMA and three,v-DMA were cytotoxic at dose levels of 100 μM –1 mM but were mutagenic only in cells expressing the metabolizing enzymes, as expected. Very similar results were obtained in a comparison of cells expressing CYP1A2 and aryl sulfotransferase. The relative contributions of aryl sulfotransferase and NAT2 to the induction of cytotoxicity and mutagenicity were compared in NER-deficient cells expressing CYP1A2 and either of the phase Ii enzymes. Aryl sulfotransferase proved to be more effective in potentiating cytotoxicity but not mutagenicity of both 2,6-DMA and 3,5-DMA.
Thus, results to date point that the alkylanilines are potent genotoxins when activated by exogenous or endogenous phase I and phase 2 xenobiotic-metabolizing enzymes. The nature of specific DNA harm products responsible for mutagenicity remains to exist identified, although evidence to date supports the suggestion that activation through North-hydroxylation and subsequent conjugation past sulfation and/or acetylation are important contributors. It is also evident that NER is an important determinant of the magnitude of mutagenic responses. Interestingly, repair deficiency also enhances cytotoxicity nether atmospheric condition in which mutagenicity is not detected, suggesting that Deoxyribonucleic acid damage other than proposed Dna adducts may as well be important in determining the observed responses.
NER of Dna is essential for maintenance of genomic integrity and cell viability. Different types of DNA lesions have distinct consequences with respect to helix distortion also as ability to suspend or obstruct DNA replication, cake ongoing transcription or hamper the battery of genome repair systems and caretakers that continuously safeguard the genome (74). Depending on the type of DNA impairment, cell death may exist triggered via apoptosis or replicative senescence (cytotoxic or cytostatic lesions, east.grand. double-strand breaks) or mutations may be induced by Dna adducts of various structural types. NER is primarily involved in repair of severely helix-distorting damage, such as bulky nucleotide adducts and intrastrand crosslinks that interfere with base pairing, thereby interfering with transcription and normal replication. Current evidence suggests that different Dna adducts may induce very unlike furnishings on jail cell proliferation, cell death via necrosis or apoptosis, too as mutagenicity, possibly owing to activation of cellular responses such every bit p53 activation.
The importance of NER status and the nature of Dna adducts as determinants of cytotoxicity and mutagenicity is well illustrated by results of a comparison of cytotoxicity and mutagenicity of a serial of carcinogens in normal diploid human fibroblasts and in cells deficient in i or more than DNA repair processes (75). Carcinogens studied included UV radiation; reactive derivatives of structurally related effluvious amides; metabolites of benzo[a]pyrene; the alkylating agent MNNG and aflatoxin B1 dichloride, a model for the reactive two,3-epoxide of aflatoxin B1. Exponentially growing cells were exposed to agents and assayed for mutations (induction of 6-thioguanine resistance) and prison cell killing (loss of colony-forming ability). Cells deficient in repair of particular Deoxyribonucleic acid adducts or lesions proved more sensitive to the agent causing those lesions than did normally repairing cells. These results are similar to our observations on alkylanilines. Many of the carcinogens were compared for their mutagenic and/or cytotoxic event not only as a office of dose administered but also equally a function of the initial number of adducts or photoproducts induced in Deoxyribonucleic acid and the number remaining at critical times posttreatment. The results demonstrated a high correlation between the number of DNA lesions remaining unexcised at the time the DNA was replicated and the frequency of mutations induced. These observations will need to exist taken into account in interpreting results of our future studies, and extensive further investigations will be required for more complete elucidation of underlying mechanisms.
Summary
Alkylanilines comprise a course of chemicals that, while clearly not every bit carcinogenic as multi-band effluvious amines, may agree far more significance for human wellness than has yet been appreciated. It appears that few individuals are spared lifelong exposure to many of these compounds. The universal nature of exposure results from an abundance of sources, some, like tobacco smoke that are well documented and others that are poorly understood but that may reverberate the widespread use of alkylanilines as components of numerous pharmaceuticals and chemical commodities. Whether their potency is sufficient to create substantial attributable risk remains an unresolved question but at that place is certainly some evidence to that consequence with regard to bladder cancer and several specific amines. Research on the mechanisms of activity of alkylanilines has even so to produce a well-defined model like the model for multi-band aromatic amines. The nowadays lack of a model may reflect more than than a lower level of interest; the mechanisms may actually be more complex ranging from nitrenium ion chemistry to reactions involving quinone imines and possibly encompassing the chemistry of ROS. The potential mechanistic complexity of alkylanilines relative to multi-ring aromatic amines is an especially important aspect of their genotoxicity. The carcinogenic multi-ring aromatic amines are accounted to target the urinary float in function because the ultimate electrophilic form, a nitrenium ion, is transported in a stable class to the bladder, where it becomes available through a change in acerbity. If additional mechanisms that practice non crave a bladder-specific environment are characteristic of alkylanilines, then there is reason to expect that they may target additional organs such as the kidney, for which there are few candidate chemical carcinogens. A improve understanding of the mechanisms of alkylaniline genotoxicity is probably to provide important insights into the environmental and genetic origins of one or more homo cancers and may reveal a substantial role for this group of compounds every bit human chemical carcinogens.
Funding
National Found of Environmental Health Sciences (PO1-ES006052; and P30-ES002109).
Acknowledgments
Conflict of Interest Statement: None declared.
Glossary
Abbreviations
| 4-ABP | 4-aminobiphenyl |
| CHO | Chinese hamster ovary |
| 2,6-DMA | 2,6-dimethylaniline |
| iii,5-DMA | three,5-dimethylaniline |
| three-EA | three-ethylaniline |
| NAT | N-acetyltransferase |
| NER | nucleotide excision repair |
| ROS | reactive oxygen species |
| TGHQ | 2,three,five-tris-(glutathion-S-yl)hydroquinone |
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