Multigenerational and Transgenerational Effects of Dioxins As the Cause of Cancer
Abstract
Dioxins are ubiquitous and persistent environmental contaminants whose background levels are still reason for concern. There is mounting evidence from both epidemiological and experimental studies that paternal exposure to the most potent congener of dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), can lower the male/female ratio of offspring. Moreover, in laboratory rodents and zebrafish, TCDD exposure of parent animals has been reported to result in reduced reproductive performance along with other adverse effects in subsequent generations, foremost through the paternal but also via the maternal germline. These impacts have been accompanied by epigenetic alterations in placenta and/or sperm cells, including changes in methylation patterns of imprinted genes. Here, we review recent key studies in this field with an attempt to provide an up-to-date picture of the present state of knowledge to the reader. These studies provide biological plausibility for the potential of dioxin exposure at a critical time-window to induce epigenetic alterations across multiple generations and the significance of aryl hydrocarbon receptor (AHR) in mediating these effects. Currently available data do not allow to accurately estimate the human health implications of these findings, although epidemiological evidence on lowered male/female ratio suggests that this effect may take place at realistic human exposure levels.
Keywords: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); dioxins; aryl hydrocarbon receptor; epigenetic modifications; transgenerational effects; gender ratio; preterm birth; paternal; maternal:
Excerpts of this article that were published online on June 17, 2019. doi: 10.3390/ijms20122947 International Journal of Molecular Sciences
http://www.mdpi.com/journal/ijms/
Abstract
Dioxins are ubiquitous and persistent environmental contaminants whose background levels are still reason for concern. There is mounting evidence from both epidemiological and experimental studies that paternal exposure to the most potent congener of dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), can lower the male/female ratio of offspring. Moreover, in laboratory rodents and zebrafish, TCDD exposure of parent animals has been reported to result in reduced reproductive performance along with other adverse effects in subsequent generations, foremost through the paternal but also via the maternal germline. These impacts have been accompanied by epigenetic alterations in placenta and/or sperm cells, including changes in methylation patterns of imprinted genes. Here, we review recent key studies in this field with an attempt to provide an up-to-date picture of the present state of knowledge to the reader. These studies provide biological plausibility for the potential of dioxin exposure at a critical time-window to induce epigenetic alterations across multiple generations and the significance of aryl hydrocarbon receptor (AHR) in mediating these effects. Currently available data do not allow to accurately estimate the human health implications of these findings, although epidemiological evidence on lowered male/female ratio suggests that this effect may take place at realistic human exposure levels.
1. Introduction
It is a well-known fact that maternal lifestyle and exposure to environmental factors during pregnancy are important for the health of the offspring. According to the developmental origins of health and disease (DOHaD) concept, environmental exposures during critical windows of development may result in functional impairment, increased risk for diseases, and other long-term health consequences later in life [1,2]. Much less attention has been paid to the toxicological significance of parental preconceptional exposures, especially effects mediated via the paternal germline [3,4]. However, there is increasingly more experimental and epidemiological evidence for potentially adverse outcomes of paternal exposures, although the current health advisories fail to emphasize the role of future fathers for the health of their offspring.
In addition to health effects of direct parental exposures on the offspring, more data are accumulating on health consequences of ancestral exposures in future generations. Understanding of the biological mechanism underlying non-genetic transgenerational inheritance of toxic effects have promoted the research of these phenomena. Paternal and transgenerational effects involve epigenetic mechanisms, and experimental studies have shown that environmentally induced epigenetic modifications of gametes may result in alterations in fertility, embryonic development, and health of the next generations [3,4,5].
As potent and persistent environmental contaminants, dioxins are still raising concerns on adverse effects on human health [6]. Due to intensive research, there are data available on the molecular mechanism of action of dioxins and their toxic effects both in experimental models and in humans. Essentially, dioxin-induced alterations have been shown be transferred to next generations predominantly via male germline after exposures during susceptibility windows of epigenetic reprogramming of primordial germ cells [7]. It is therefore worthwhile to include dioxins in further attempts to elucidate the characteristics and significance of epigenetically mediated transgenerational effects. In a recent minireview, Brehm and Flaws [8] summarized the transgenerational effects of a whole range of different types of endocrine disrupting chemicals on male and female reproduction. The purpose of this mini review is to focus on available data on paternally and maternally mediated multi- and transgenerational effects of dioxins.
1.1. Epigenetic Alterations and Environmental Factors
Epigenetics involves a variety of mechanisms that can regulate gene expression without alterations in the underlying DNA sequence in the genome. These include DNA methylation, histone modifications, non-coding RNAs, chromatin structure, and RNA methylations [5] (Figure 1). Epigenetic modifications play a significant role in normal development and they also allow organisms to adapt into a changing environment. However, altered gene expression and consequently altered phenotype may also result in toxic effects or disease states. From a toxicological point of view, an important property of epigenetic alterations in gametes is that when they occur in imprinted genes, they can be transmitted to subsequent generations. Imprinted genes are methylated in either the female (maternally imprinted) or the male (paternally imprinted) germline, and they retain this inheritance pattern in the next generation. In mammals, some 150 imprinted genes have been identified to date [9].
Previous studies have shown that exposure to different types of environmental and nutritional factors, such as chemicals, high-fat diet, caloric restriction, and stress during embryogenesis may result in adult-onset toxic effects or disease states for multiple generations [5,10,11,12]. These effects are called multigenerational when there is a direct exposure of the generation to the environmental factor, and in contrast, if the effects are transmitted in the germ line without continued involvement of direct exposure, they are transgenerational (also called ancestral exposure). If a pregnant female is exposed, there is a direct exposure of three generations: The female (F0 generation), the fetuses (F1 generation) and the germline of the fetuses (prospective F2 generation). Therefore, the F3 generation is the first unexposed generation, and effects observed in the F3 and subsequent generations are transgenerational. On the other hand, if a male or preconception female (F0) is exposed, also the germline (F1, eggs or sperm) is directly exposed, and in this case F2 generation is the first unexposed generation. Similarly, in fish and other species with external fertilization and embryonic development, exposure of parent animals (F0) involves also exposure of the F1 germline, and F2 generation is the first unexposed generation.
Upon fertilization, there is a rapid demethylation of most of the paternal (and slower for maternal) genome by the blastocyst stage, although e.g., imprinted genes show resistance. Soon after implantation, a wave of global de novo methylation occurs and is maintained in somatic cells. In primordial germ cells, however, another demethylation step (more substantial than the first one) follows, reaching its peak at embryonic days 11.5–12.5 in mice. In the male germline, de novo methylation is then soon initiated (at embryonic day 13.5 in mice), and the male methylome is completely established prior to birth. In contrast, de novo methylation does not begin until after birth in the female germline, reaching completion by postnatal day 21 in mice [13,14].
1.2. Dioxins
“Dioxins” is the common name for a large number of potent and persistent environmental pollutants that include polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (DL-PCBs) (Figure 2). PCDDs and PCDFs are formed as unwanted by-products in waste incineration at low temperatures and in industrial processes, but PCBs have been intentionally manufactured for a variety of industrial applications between 1929 and 1970s when they were banned. Due to the persistence and ability of dioxins to biomagnify in the food chain, humans are still exposed to them mainly via food. The most potent congener of dioxins is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which therefore serves as the toxicological prototype for the entire group. Dioxins are presumed to share a common mode of action and to display a purely additional co-effect (dose additivity). To facilitate the assessment of toxicity of dioxin mixtures, all dioxin congeners have been assigned a toxic equivalency factor (TEF) relative to TCDD (which has the TEF of 1). When the concentration of each congener in the mixture is multiplied by its TEF value and the resultant products are summed up, the result shows the amount of TCDD the mixture equals to in terms of its toxicity (TEQ).
5. Conclusions and Future Prospects
In humans, rodents and zebrafish, paternal (or paternal and maternal) exposure to TCDD has been reported to result in a lowered male/female ratio in offspring. Additional in vitro studies on mouse sperm suggest that this could be at least partly due to TCDD-induced reduction in survival and fertilization capability of spermatozoa carrying the Y chromosome. Male mice exposed perinatally to TCDD and mated with unexposed females have diminished fertility and the gestational length of their partners is shortened, possibly because of lowered PGR but elevated TLR4 abundance (shown for their mRNAs) in placenta. Similarly, in utero and lactational exposure to TCDD of either male or female F1 mice can reduce fertility and the length of gestation at least up to F3 generation. These phenomena are associated with a decreased abundance of PGR isoforms A and B in uterus in the case of maternal germline and increased AHR expression in spermatocytes for paternal germline. In the latter case, a hyper-inflammatory testicular phenotype has also been recorded. Moreover, paternally mediated effects have been found to be accompanied by a large number of differentially methylated regions in the DNA of sperm and placenta.
In rats, paternal germline has exhibited a decreased proportion of implantations per corpus luteum after TCDD exposure. In an experimental setting where descendants of dioxin exposed F0 rat dams are mated with one another, a number of abnormalities have been noticed in the F3 generation, including elevated total disease incidence and a reduced number of ovarian primordial follicles.
In zebrafish, egg release and egg fertilization have proven to be transgenerationally impaired after TCDD exposure. These studies also reported skeletal effects of TCDD up to the F2 generation.
Most of the studies assessing the mode of inheritance of TCDD-induced transgenerational impacts have so far assessed paternal germline effects alone. In those cases where these have been contrasted with maternal effects (in mice and zebrafish), the outcomes have proven to be qualitatively similar. However, at least in zebrafish, the magnitude of the effects was larger in the case of paternal germline.
The available data provide the proof of principle on the potential of dioxins to induce epigenetic alterations and related toxic effects across multiple generations. On the other hand, the significance of these findings for human health is difficult to assess as there is too little information at present on the dose-responses of the multi- and transgenerational effects of TCDD. At present, it appears that these phenomena require fairly high exposures (close to the doses causing teratogenic manifestations). The available rodent studies have been carried out at dose levels between 0.1 and 10 µg/kg bw that are associated with dioxin body burdens clearly above the current human background exposure levels [6,125]. The most sensitive effect reported so far was the decreased proportion of implantations per corpus luteum in the rat offspring of F2 and F3 generations at 0.1 µg/kg bw [117]. However, it is important to note that the human studies on lowered male/female ratio in the offspring of TCDD exposed fathers suggest that this effect may take place at relatively low exposure level, starting at serum TCDD concentrations >15 pg/g fat [98] or ≥20 pg/g fat [100].
Transgenerational impacts of TCDD have emerged in conjunction with cellular epigenetic modifications, including altered methylation patterns of imprinted genes. Verifying whether this is causal relationship and pinpointing the key genes thus affected require further studies in the future. Likewise, the indispensable role of the AHR in these phenomena awaits formal verification using AHR-deficient animals. The molecular mechanisms upon AHR binding that lead to epigenetic alterations and the capability of other AHR agonists to cause them and transgenerational health effects will be further important research topics. Finally, with regard to the DoHaD concept the human health significance of the dioxin-induced preconceptional paternally mediated and transgenerational effects should be addressed in future studies.