AHR-mediated effects

AHR has been recognised for decades as a ligand-activated transcription factor that is—along with its nuclear translocator—responsible for the induction of drug metabolising enzymes such as cytochrome P450-dependent monooxygenases. Not until recently have other functions of this protein begun to be recognised, and it is now clear that AHR also functions in pathways beyond xenobiotic metabolising enzyme induction. Chemical impairment of these other pathways may help to explain some aspects of compound-inherent toxicities. Ligand-dependent activation of AHR may thus affect mitogen-activated protein kinase cascades or other cellular kinases, cell cycle progression, differentiation and apoptosis. Ultimately, the effect of a particular AHR ligand on the biology of the organism will depend on the milieu of critical pathways and proteins expressed in specific cells and tissues with which AHR itself interacts.2

The unintentionally generated environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; ‘dioxin’) is classified as a strong human carcinogen. This compound is known as a potent non-genotoxic tumour promoter in liver, thyroid, lung, adrenal cortex, lymph nodes and skin and is one of the strongest agonists of AHR as yet characterised.3 Besides its tumorigenicity, TCDD induces a great diversity of AHR-mediated toxic effects in vivo without requiring metabolic activation ahead of its action. In recent years it became clear that TCDD changes the expression levels of numerous proteins involved in cell and tissue homeostasis, ie, cellular proliferation (T_H17 cells), cell cycle regulation and apoptosis, or extracellular matrix turnover, cytokine production, signalling and cell adhesion. In addition to their transactivation activity AHR ligand complexes also affect cellular signalling networks such as those triggered by hormones (eg, oestrogen receptor beta), hypoxia (hypoxia-inducible factor 1 alpha), nuclear factor kappa B (NF-κB), retinoblastoma protein, or by protein kinases, phosphatases and their coactivators through molecular crosstalk or by epigenetic signalling (further discussed below). At present, the individual roles of most of these AHR-mediated effects in TCDD-induced carcinogenesis remain elusive and thus need to be addressed further. Certainly, all compound-induced alterations together contribute to a complex network of gene–gene interactions that may help to explain the broad-ranging interactive and tissue-specific biological outcomes observed upon exposure to TCDD or other AHR agonists.

Disruption of endocrine signalling

Endocrine-disrupting chemicals (EDC) have been recognised as a major environmental and putative human health problem mainly by two lines of evidence. First, observations from sensitive ecosystems indicated that adverse hormonal effects might be triggered by various environmental pollutants, such as, for example, polychlorinated biphenyls, DDT-like or alkylated tin compounds.4 Second, covering the past few decades human epidemiological studies demonstrated an increase in several hormone-dependent cancers (eg, breast carcinoma), or affected fertility markers such as decreasing overall sperm counts in men. Nevertheless, reliable causal associations between such observations and the exposure to EDC have only been established for a few examples in humans so far. One of the best worked out examples is the oestrogenic agonist diethylstilbestrol, which was used until the 1970s to prevent miscarriages, and was later recognised to promote rare types of vaginal tumours. These tumours occurred in the adult daughters of women who had been treated more than a decade earlier. Among EDC, oestrogen-active substances constitute a major concern.5 Up to 200 compounds have been identified as being capable of binding and activating oestrogen receptor alpha and/or oestrogen receptor β. The receptor affinity of these agonists is usually 10⁵–10⁸ times lower compared with diethylstilbestrol, or with the most important physiological substrate of oestradiol. Nevertheless, some synthetic substances widely used as pesticides, plasticizers or as additives in food-packaging materials raised concern due to their presence in the food chain at low but chronic levels. The relationships between such exposures and adverse health effects in humans remain to be investigated further by integrating epidemiological and toxicological studies.

EDC constitute a chemically diverse group of substances that had been shown to alter various hormonal functions in vivo. Although this might affect reproduction or development, the term EDC is often used in an ambiguous way, and effects on development, on reproduction or on body metabolism and homeostasis are often not well separated from each other. EDC can adversely affect multiple hormone-receptor systems or act in a receptor-independent way. Consequently, a whole range of different adverse effects can be anticipated; many of them address multiple hormone-sensitive systems simultaneously.5 For example, the earlier onset of female puberty, increased sensitivity of adipose tissue towards insulin, obesity, and neuronal developmental damage had previously been linked to various EDC; these effects urgently require further clarification. Selected EDC, including pesticides, as well as consumer-relevant compounds such as bisphenol A or nonylphenols are now well characterised (and regulated in some countries); however, the overall risk resulting from EDC is still difficult to assess. Both the US Environmental Protection Agency6 and the European Commission7 had initiated screening programmes in the late 1990s, aimed at identifying EDC for further characterisation, according to prioritisation criteria. It still remains a major challenge to develop and validate test methods that are suited to cover the entire functional spectrum of potential EDC, thereby providing a meaningful guidance for the quantification of risk at all levels of complexity to be assessed; that is, tissue and organ-specific toxicities, adversities on whole organisms including humans, and the overall impact on human environments and the biosphere.

Reactive oxygen species

Reactive oxygen species (ROS) are generated as by-products of mitochondrial respiration and by other enzymatic oxidations, which are predominantly associated with peroxisomes and microsomes. ROS include three major species of reduced oxygen: superoxide anion, hydrogen peroxide and hydroxy radicals. Excessive generation of ROS, as for example in chronic inflammatory diseases, or after exposure to redox-active metals, can trigger oxidative stress, a metabolic condition associated with adverse molecular effects such as lipid peroxidation and oxidative DNA damage. In addition to its deleterious effects on cells and tissues in general, endogenous ROS also play an important role in the cellular immune response.8 So, hydrogen peroxide and superoxide have been identified as second messengers that are generated after the activation of various cytokine receptors, including interleukin receptor 1, Toll-like receptor 4 and tumour necrosis factor alpha receptor 1.9

One central mechanism that affects the levels of endogenous ROS is controlled by the peroxisome proliferator-activated receptor (PPAR), which activates the expression of various peroxisomal and microsomal enzymes involved in the degradation of complex fatty acids and accompanying generation of hydrogen peroxide. Besides activation by endogenous lipids, PPARα can also be activated by xenobiotics, thereby enhancing the formation of ROS, and consequently executing the disastrous effects of non-genotoxic carcinogens among this group of chemicals.10 Examples of exogenous PPAR activators include phthalate monoesters and fibrate drugs.

ROS-associated mutagenesis is predominately triggered by hydroxy radicals, known to form preferentially 8-hydroxy 2′-desoxyguanyl residues in DNA.11 Hydroxy radicals arise by the further reduction of hydrogen peroxide in Fenton's reactions, dependent on superoxide anion or redox-active metals. This might provide another important link between ROS and the toxic effects of dietary and environmental pollutants. In fact, the induction of oxidative stress is often discussed as a putative carcinogenic mechanism for various metals, including arsenic, chromium or nickel, and for organic xenobiotics such as dioxins (ie, TCDD) or benzopyrenes.12 In addition, ROS have also been shown to activate mitogen-activated protein kinases and to trigger signalling pathways, notably NF-κB and activator protein 1. ROS-mediated proliferation could facilitate an accelerated accumulation of mutations, initially triggered by oxidative DNA damage, and thus contribute to genetic instability and to the so-called mutator phenotype of cancer cells proposed some time ago.13 In fact, xenobiotic compounds that generate ROS can also frequently act as powerful tumour promoters.

In general, it remains difficult to sort out the specific contribution and significance of ROS in carcinogenesis (and other diseases), because various alternative mechanisms—such as the formation of bulky DNA adducts, inhibition of DNA repair enzymes or induction of epigenetic effects—apply in parallel for most of the known carcinogenic metals or organic compounds.12

Apoptotic resistance

It is well known from chemotherapy that exposure to genotoxic drugs can induce resistance towards apoptosis in the cells treated. One important mechanism involves the inactivation of tumour suppressor proteins, especially TP53 by mutations. In addition, non-genotoxic mechanisms leading to enhanced activation of certain signalling pathways such as phosphatidylinositol 3 kinase-Akt or NF-κB have been found to confer apoptosis resistance in experimental models.14 Target genes of NF-κB include various caspase inhibitors, such as IAP and c-FLIP, as well as Bcl-2 homologues (Bcl-XL) and TRAF2.14 15 Although some environmental xenobiotics and carcinogenic metals activate NF-κB by the generation of ROS, until now implications for apoptosis resistance have hardly been explored; such implications certainly need to be worked out in the years to come, in particular regarding the relation to the adverse inflammatory or carcinogenic effects of environmental toxins.

Epigenetic effects

Effects of xenobiotics on the epigenome are very complex. As an important example, activation of the AHR/arylhydrocarbon receptor nuclear translocator involves binding to histone modifying co-factors that modulate the expression of target genes.16 Signalling cross-talk between AHR and other pathways, especially NF-κB, was further shown to be regulated at the epigenetic level, affecting both the methylation and acetylation of histones.16 In general, however, the molecular details of this regulation—and the precise consequences for carcinogenesis, inflammation or other adverse effects triggered by exogenous AHR activators—are far from being fully understood. Other xenobiotics, especially metalloid compounds, were shown to affect DNA methylation and promoter activity without interfering with AHR. Again, the mechanisms remain to be clarified in detail. As an example, the tumour suppressor protein p16^INK4a is inactivated by hypermethylation after exposure to various metals, including arsenic and nickel.12 This observation might reflect the outcome of a common mode depending on the intermediate generation of ROS. Intriguingly, arsenic was also shown to trigger the hypomethylation of DNA, thereby activating the expression of cyclin D1 and oestrogen receptor alpha.12 These different effects on methylation patterns of individual genes are difficult to reconcile, but might indicate pleiotropic interactions between xenobiotic compounds and epigenetic regulation that are responsible for the varying and complex outcomes observed. The overall relevance for metal-induced carcinogenesis or other implications for human health remain elusive.