Health Effects of Dioxin: Dr. Coral Wynter study Part II

New Discoveries of the Health Effects of Dioxin, the Active Agent in Agent Orange 

By Coral Wynter (PhD Lond.)

The health effects of exposure of humans to dioxin, 2,3,7 8 tetrachloro dibenzo dioxin, or TCDD, the active agent in Agent Orange, which was used extensively in the Vietnam War, have been well documented (see earlier article in links.org.au/node/3636). However the latest research shows that dioxin belongs to a group of chemicals that can cause permanent damage to the human genome, called Epigenetic Transgenerational Inheritance. This article will try to explain what that means and the frightening consequences. 

The first visible effect of dioxin is skin lesions, such as chloracne, leaving pustules and scars. Chloracne is a severe skin disease with acne like lesions as evident in the face of Viktor Yushchenko in 2004, a victim of dioxin poisoning and vicious Ukraine politics even then (Fig. 1). The toxic effects appear to be dose-dependent but even very tiny amounts of the chemical, in parts per trillion, can have an effect. Chloracne is a skin disease, often accompanied by severe disfiguration, severe joint pain, headaches, fatigue, irritability and chronic weakness; and it can persist in the body for at least 30 years after exposure (Kimbrough & Grandjean, 1988). Chloracne occurs mainly on the face and upper body and other effects are discoloration of the skin and excessive body hair. Changes in blood and urine composition may also indicate liver damage as well. One of the persistent effects, after the initial illness, is also amenorrhea, the absence of menstrual period in women of a fertile age. Exposure to high concentrations of dioxin may induce long term alterations in glucose metabolism and subtle changes in hormonal levels due to the disruption of the endocrine system (WHO Fact sheet No 225, May 2010). 

Fig.1 Viktor Yushchenko, Ukrainian politician,
after he was poisoned with dioxin in 2004
(Sorg et al 2009). 

TCDD also weakens the immune system and causes a decrease in the body’s ability to fight bacteria and viruses, thus causing a greater susceptibility to infections. Other diseases linked to dioxin are amyloidosis (abnormal protein deposits), Parkinson’s disease, porphyria cutanea tarda (a blood and skin disorder), ischemic heart disease, hypertension, Type 2 diabetes, peripheral neuropathy (the deadening of the nerves in the extremities such as toes and fingers) and lipid metabolism disorders. Other suspected effects in adults besides liver damage, are alterations in heme metabolism (heme proteins carry oxygen in red blood cells), serum lipid levels, and thyroid functions. Actually, thyroid status in particular is a sensitive marker of the degree of exposure to dioxin. 

The US Department of Health and Human Services has determined that TCDD may reasonably be anticipated to cause cancer. US Veterans serving in the South of Vietnam had higher rates of throat cancer, acute/chronic leukemia, Hodgkin’s lymphoma, (a cancer originating from white blood cells) and non-Hodgkin's lymphoma, chronic lymphocytic leukemia (including hairy-cell leukemia), multiple myeloma, prostate cancer, lung cancer (including bronchea or trachea), colon cancer, softtissue sarcoma and liver cancer. Dioxin and similar compounds have also been shown not to have any direct mutagenic or genotoxic activity. Their main action in causing cancer is cancer promotion. 

One of the most worrying effects is the discovery that dioxin poisoning affects brain function, associated with neurobehavioral abnormalities, including cognitive and locomotor systems. While the specific brain cell types have been difficult to identify, the abnormalities may be consistent with findings of improper brain maturation. Animal studies have shown that perinatal exposure to TCDD has a marked effect on learning ability in rat and monkeys (Negishi et al 2006). Epidemiological studies have shown that children accidently exposed to Polychlorinated Biphenyls (PCBs) and TCDD show delayed motor development and a tendency to hyperactivity (Hazel et al 2006). 

The developmental effects on the foetus if the mother has been exposed to dioxin through the food chain or water supplies may be much more important than the effects in adults. Dioxin promotes teratogenicity, (birth defects) and congenital defects in the off-spring, including such problems as cleft lip, cleft palate, club foot, hydrocephalus, neural tube defects, fused fingers, muscle malformations and paralysis, kidney damage, Spina Bifida, and some developmental disabilities. These include disturbances of tooth development, and of sexual development. Other effects on the developing foetus include again impairment of the immune system, liver damage, heart problems, wasting disease, skeletal deformities, changes in endocrine homeostasis (the endocrine glands release hormones in responses to stress and injury), growth and development, absorption of nutrients, energy metabolism, water and electrolyte balance, reproduction, birth, and lactation, reduction in steroid-dependent response, modification of cell growth, growth of tissues and differentiation of tissues (specialisation). An Australian study found some evidence of increased heart defects and Down's Syndrome in babies born to mothers exposed to dioxin.

In Vietnam, because the whole environment was contaminated with Agent Orange, husbands and wives were both exposed to TCDD. Several effects on pregnancy and birth, including hydatidiform mole, are thought to be linked to dioxin exposure. Hydatidiform mole is an abnormal pregnancy in which placental tissue grows in an uncontrolled way and the embryo does not develop. It carries a risk of cancer which can be fatal without intensive chemotherapy. This list covers virtually every organ and every function of the body.

We may not think this affects us, if we are not living in South Vietnam, which is still heavily contaminated. But it does because everyone living in industrialized countries has a potent mixture of dioxins, furans, co-planar Poly-Chlorinated Biphenyls, Poly-Chlorinated Naphthalenes and other similar compounds stored and accumulated in their body fat. 

Dioxins occur as by-products in the manufacture of some organochlorines, in the incineration of chlorine-containing substances such as polyvinyl chloride, in the chlorine bleaching of paper, and from natural sources such as volcanoes and forest fires. Ninety per cent of people's overall exposure to dioxins is estimated to be from the diet. Meat, milk products and fish have higher levels of dioxins and furans than fruit, vegetables and grains. Probably the only way to avoid eating or drinking these chemicals is to adopt a vegan diet. Things are so bad, Greenpeace is calling for the cessation of all industrial chlorine production. 

Mechanism of Action of Dioxin in the Cell

The simplest way to explain one of the toxic effects of dioxin is that it binds to a transcription factor found in the cytoplasm of the cell, called the Aryl hydrocarbon Receptor or AhR (Fig. 2). The role of DNA and RNA in protein synthesis is a process known as DNA Transcription and Translation. In transcription, only one strand of the DNA is transcribed into Messenger RNA (mRNA). In translation the mRNA is translated into a polypeptide chain. This polypeptide chain is then folded into a unique protein used in a particular cell function. AhR is an extremely important transcription factor which when bound to dioxin moves into the cell nucleus and switches on and off a whole battery of genes (Denison et al 2011). Thus dioxin has no mutation effect nor can it cause breakage of chromosomes but it modifies many of the controlling elements for transcription of target genes into messenger RNA (mRNA). It thus alters the timing of the entire normal functioning of the cell’s metabolism.

Fig. 2
Description of AhR signalling in mammals and
its interaction with dioxin.

TCDD enters the cell by diffusion across the cell membrane, which is composed of lipids. Normally a water-soluble chemical would require a specific transporter across the cell membrane, but the dioxins diffuse across the cell membrane because of their lipid solubility (hydrophobic). AhR exists in an inactive state as a multiprotein complex in the cytoplasm. When the dioxin molecule binds to the AhR multiprotein complex, it undergoes a conformational change (changes its shape). This causes AhR to move into the cell nucleus, where the chromosomes and most of the cell’s DNA is contained. Once in the nucleus, AhR with its dioxin load binds to a structurally related protein called ARNT or Ah Receptor Nuclear Translocator. This results in the transformation of the AhR:ARNT:dioxin complex into a structure which can bind DNA with very high affinity. The DNA recognition site referred to as a dioxin-responsible element (DRE) upstream of the section of DNA coding for a gene. This leads to increased rates of gene transcription into mRNA and then protein. So the entire regulated control of switching on certain genes is reorganised and the result is metabolic chaos.

The toxicity of dioxins also results from their ability to persistently activate or repress expression of key AhR responsible genes. The extent of the effect on gene expression can be demonstrated from microarray analysis of DNA from mouse cells in which the mouse AhR molecule was responsible for binding to 1,752 genes, the DRE recognition sites. A similar number of genes would be expected to respond in humans but this work has not yet been published. In relative terms this is an enormous number of our total gene pool, around 10%. So it is no wonder that a large number of cellular metabolic processes are in a shambles.

Not every target of the AhR:ARNT:TCDD system can be directly linked to the specific genes causing each one of the observed health effects but these are topics of ongoing investigations. One of the first systems to be studied was the enzyme cytochrome P450 1A1 or CYP1A1, which is a carcinogen-activating enzyme that catalyzes the metabolic activation of several procarcinogens to their ultimate carcinogenic forms. The activity of CYP1A1 is increased by dioxin to very high levels. Moreover, several studies have demonstrated the positive relationship between the induction of CYP1A1 and the incidence of several human cancers such as lung, colon, and rectal cancers (Oyama et al 2007; Slattery et al 2004.)

One of the most worrying effects is the discovery that dioxin poisoning affects brain function, associated with neurobehavioral abnormalities, including cognitive and locomotor systems. While the specific brain cell types have been difficult to identify, the abnormalities may be consistent with findings of improper brain maturation. Epidemiological studies have shown that children accidently exposed to PCBs and TCDD show delayed motor development and a tendency to hyperactivity (Hazel et al 2006).

Another report has indicated an association of serum concentrations of dioxins with the prevalence of learning disability and attention deficit hyperactivity disorder (ADHD). However in a recent paper, it was demonstrated that mice neuroblastoma cells exhibited over expression of and functional activity of AhR multiprotein complex. Now tyrosine hydroxylase is a rate-limiting enzyme for dopamine synthesis. Behavioural abnormalities including ADHD are thought to be closely related to dopaminergic neural function. This means that if there is an increasing burden of TCDDs and PCBs in our food chain, an increasing number of children exposed pre-natal or during early development will have increasing cognitive disabilities. This could help explain the huge increase in ADHD amongst present-day school children. Suppression of liver gluconeogenesis, associated with wasting disease, loss of immune response, changes in cell plasticity, cytosketelal reorganization, increased cell migration are based on this disruption of the AhR normal function. There are now hundreds of scientific papers providing a detailed mechanism of how the normal development and metabolism of humans is affected after AhR is bound to TCDD.

Methylation of DNA

The most worrying and disturbing effects of dioxin are due to its permanent remodelling of the human genome and how this will affect the future generations of children of those victims who have been exposed to dioxin. The biological mechanism of this effect has only come to light in the last 5 years as we have begun to understand the different mechanisms controlling expression of the genome in each cell. To explain this process, some background information about how DNA is bundled up in the cell and how it is switched on, is necessary.

The DNA is the actual blueprint for each human being and is different for each individual. The DNA consists of four nucleotides in a long chain, guanine, adenine, thymine and cytosine, referred to as AGTC. Every cell in the body contains the same DNA in the cell nucleus as every other cell, in the form of two lots of 22 chromosomes, one from the mother and another set from the father, as well as 2 X chromosomes if female and an X and Y chromosome if male, giving a total of 46 chromosomes. However the gonads, ie the ovary and the sperm cells, contain only half the DNA, 23 chromosomes. The sequence of the 3 billion nucleotides of the DNA in the 23 chromosomes was funded by a joint British and US government project, known as the Human Genome Project, in 2000, and in a completed form in 2003 for one individual. There are only 30-40,000 genes that are translated into proteins, far fewer than the expected 100,000 genes, and only twice as many as in a fruit fly. The genes for translated proteins make up only 1.5% of the genome, the rest once thought to be junk DNA controls the mechanism to determine when and how the genes are switched on, providing the organisation to build tissue and organs.

Another significant factor of DNA is that one of the nucleotides can be methylated, that is cytosine can be converted to 5’methyl cytosine, next to a guanine called a CpG site. If this methylation occurs upstream of the promoter site, which is responsible for switching on the gene, then the gene will be silenced and no translation occurs. If the silenced gene is a gene responsible for suppressing the growth of tumours, then the resulting loss in translation will result in a cancer, an uncontrollable growth of cells. A methylated cytosine nucleotide is now considered so important due to its role in switching genes on and off, that it is called the fifth base (Wynter et al 2007a). In vertebrates about 30% of all genes contain a CpG island. The rest of the genes are silenced by a different mechanism, often unknown. About 70% of all CpG sites in the genome are methylated in normal human cells as it is the main mechanism for shutting down unwanted translation of bits of foreign genes, repetitive DNA and satellite DNA.

The majority of DNA sequences in the cell nucleus exist in a very condensed form called heterochromatin, rather like skeins of wool (Wynter et al 2007b) The vast majority of the DNA is unavailable for transcription, that is, translation into mRNA and ultimately a protein. The basic unit of the chromatin is a nucleosome, which is a DNA chain of 146 nucleotides wrapped around a core of eight histones, a special protein which has an overall negative charge. The state of heterochromatin is very dynamic, controlled by an army of 100 different proteins and enzymes that modify the histones to enable the DNA to be exposed for transcription or buried, never to see the light of day (Fig. 3).

So it has only recently been understood that there are two levels at which the genome can be affected 1) through a mutagen causing mutation, which directly changes the sequence of nucleotides or 2) at the level of the modifications of the nucleotides, methylation of cytosine (CpG) or chemical modifications of the proteins surrounding the genome which in turn modifies the ability to read out the transcription of the genome correctly, both temporally and quantitatively. This second finely-turned level of control of DNA transcription is called epigenetics, from the Greek meaning ‘on top of genetics.’ This is how the environment influences growth, development and health. Methylation and epigenetics is the reason identical twins sometimes look quite different, due to a different response to the environment. In other words, it is a system in addition to the direct translation of the genetic information that switches the genetic information on and off. Dioxin is only a very weak mutagen but it can change the epigenome and hence it affects the following generations, who have had no exposure to dioxin.

This topic of research is epigenetic transgenerational inheritance and involves the germline transmission of an altered epigenome and phenotype [across generations in the absence of any direct environmental exposure]. This is why dioxin is so dangerous as it permanently changes the epigenome of subsequent generations. To understand how this occurs we need to know about the formation of gonadal sex determination and what happens in the gonads, the ova (female eggs) and the formation of sperm and the zygote (the union of sperm and ovum).

Fig. 3 Transcriptional regulation at the epigenetic level. DNA methylation at CpG sites usually suppresses gene expression. Shown is a cytosine nucleotide, with a methyl group attached. Histones are globular proteins that undergo post-translational modifications. Active genes are usually characterized by low DNA methylation and highly acetylated chromatin configuration that allow access to transcription factors (Hou et al 2012).

The Role of Epigenetics in the Formation of Gametes 

During the process of fertilisation which takes place in the fallopian tubes, the sperm enters the ovum, the egg cell, with its nucleus now called a pronucleus, containing the haploid number of DNA, 23 chromosomes (Fig. 4). The female pronucleus is also carrying the haploid number of DNA. The pronuclei do not actually fuse, but the membranes around the pronuclei dissolve and their chromosomes combine, now becoming part of a single diploid nucleus in the resulting embryo, called a zygote. This zygote has the developmental potential to generate an entire organism, about 50 trillion cells. All cells descended from this zygote share the exact DNA sequence of 3 billion nucleotides in each cell. Each cell of the preimplantation embryo is called totipotent. That is, each cell has the potential to form all of the different cell types in the developing embryo. This totipotency means that some cells can be removed from the preimplantation embryo and the remaining cells will compensate for their absence. This has allowed the development of a technique known as preimplantation genetic diagnosis (PGD), whereby a small number of cells from the preimplantation embryo created by IVF, can be removed by biopsy, analysed genetically to detect any mutations and selected for implantation in the uterus. The embryo attaches to the endometrium of the uterus at 7 days.

Fig. 4 Human zygote showing both male and female pronuclei before fusion. B) Zygote after cell division developing into a blastula and gastrula.

Because the methylation on the cytosine blocks transcription, this block has to be removed in order to allow the zygote to develop. A wave of demethylation occurs in the sperm pronucleus, which is followed by a passive wave of demethylation in the female pronucleus. In other words, this dramatic change in the epigenetic status allows the zygote to erase the epigenetic signature inherited from the gametes and to begin the growth and development of the new organism (Seisenberger et al 2013).

Gastrulation is an early phase in the development of the embryo during which the single-layered blastula is reorganised into a three-layered structure, known as the gastrula. These 3 germ layers are known as the ectoderm, mesoderm and endoderm. At the time of gastrulation a small group of cells are "put aside" to later form oocytes and spermatozoa. This population of cells is described as the primordial germ cells (PGCs) and are already formed in the second week of the fertilised human embryo. These cells also migrate initially into the posterior endoderm that forms the hindgut and from there into the genital ridge that will be the site of the developing gonad. Lewis Wolpert, pioneering developmental biologist and a bit of a wit, noted that, "It is not birth, marriage, or death, but gastrulation, which is truly the most important time in your life."

As a consequence, the PGCs need to reprogram this inherited somatic epigenetic pattern if it is to give rise to the gametes to form the next generation. That is the second generation of humans both male and female are formed at this very early stage in the human embryo. A wave of demethylation occurs, a resetting of the chromatin structure in order to fulfil its role as sperm or ova. This re-establishes pluripotency. The parental methylation imprints are erased in PCGs and new imprints are established to reflect the gender of the embryo. These imprints are then maintained in the gametes derived from the PGCs. Following demethylation in the early PCGs, the genome must undergo de novo (new) methylation in order to achieve the much higher levels of methylation found in mature gametes. In female germ cells, the DNA methylation establishment takes place after birth in the growing oocyte. In male PGCs new methylation takes place several days after erasure is completed. It appears that PCGs are the main sites for DNA methylation reprogramming during the mammalian life cycle.

Proliferation of the female germ cells only takes place during embryogenesis in contrast to the continuous proliferation of male germ cells. It has long been held as a dogma that the ovary has a finite number of primordial follicles, which together with the rate of depletion of this pool, determines the female reproductive life span. There has been a recent report of germline stem cells in the ovary. This pool of stem cells may replenish the ovary with new oocytes, during adult life, but this has yet to be confirmed (Bhartiya et al 2012).

Fig. 5 The primordial germ cells (PGC, red dots, 6) wander out of the yolk sac wall, along the zona pellucida (2) and the dorsal mesentery, into the gonadal ridge (5). In humans, the PGC reach this structure in the 6th week. Number 1 is the rectum, no 3 is the allantois (collects liquid waste from the embryo), no 4 is the nephrogenic cord (giving rise to the kidneys and urethers), and no 7 is the heart lump.

Another huge suppression event is the epigenetic system which enables men and women to have equal expression of the genes carried on the X chromosome, despite the fact that women have two X chromosome copies and men have only one X (in addition to a Y chromosome). The system is called X chromosome inactivation. This cellular system ensures that only one copy of the X chromosomal genes in a female is ‘active’. Most of the other genes on the other partner X chromosome copy are ‘inactivated’ or switched off. In any one individual, this is a totally random event with some cells having the X chromosome that came from their mother switched off (an inactive maternal X chromosome); other cells will have the paternal X chromosome inactivated.

All this is further complicated by the fact that during the processes of demethylation and re-methylation, some parts of the genome strictly maintain their original methylation status. Some genes are differentially marked by DNA methylation so that the two different copies of the same gene, one from the female and one from the male, are marked so the cell can differentiate which is which and switches on only one copy of the gene in the two similar chromosomes. This leads to parent-of-origin specific expression. They are called imprinted genes and play an important role in regulating growth in the embryo and post-natal development as well as behaviour. Some diseases caused by wrong parental imprinting are the Angelman syndrome, Prader-Willi syndrome, Russell-Silver syndrome and the Beckwith-Wiedemann syndromes. Famous Scottish crime writer Ian Rankin has a son with Angelman syndrome. However these imprinted marks are erased during formation of PCGs and new imprints are established that reflect the gender of the embryo.

Another function of methylation is to silence enormously long arrays of unwanted and unusable DNA. The human genome contains transposable elements (called TEs) that can move around within the chromosomes, which also incidentally allow for genetic innovation as well as disease and are sometimes called ‘jumping genes.’ Almost half of our genome is derived from TEs (Cordaux and Batzer 2009). Considering that protein coding regions account for just 1.5% of the human genome, this is incredible. TEs can be separated into 2 major classes, transposons and retrotransposons (Fig. 5). DNA transposons make up 3% of the human genome. Retrotransposons replicate via RNA copied into DNA and then insert themselves into the genome. Retrotransposons in turn can be divided into two classes, those that have Long Terminal Repeats (called LTRs), these are endogenous retroviruses accounting for 8% of the genome. The vast majority of human TEs are non-LTR retrotransposons, typified by LINE-1, Alu and SVA elements and account for 30% of the genome. These last 3 groups are currently active in the human genome. These non-LTR retrotransposons can also generate microsatellites, markers which are very useful in identifying individuals and are used by the FBI, and police forces all over the world. The retrotransposons have dramatically impacted human evolution over the last 37 million years (Cordaux and Batzer 2009).

Fig. 6 Composition of the human genome.

Finally it can be seen that the methylation of DNA is involved in all of these processes; parental imprinting, inactivation of the second X chromosome, control of the transposable elements, which can cause a long list of diseases. Methylation is essential for genetic stability of the genome and its transmission to the next generation. Thus any disruption of this DNA methylation or epigenome can spell disaster for the offspring. The complex factors that control methylation and essentially protect some regions of the genome and allow other regions to be remethylated are still totally unknown. Any interference in this process usually produces a deformed individual or if a healthy baby is born, later on in life he/she will succumb to an early death. This complexity of DNA methylation accounts for the fact that it has been extremely difficult to clone any animal and not have it die early from a disease.

Thus genetically induced germline epigenetic modifications can occur during this DNA demethylation and remethylation period during fetal development and become permanently reprogrammed (Skinner et al 2010). The male germline propagates these epigenetic changes after the fertilization to all somatic cells resulting in an altered epigenome that can lead to adult onset disease in future generations. The female ovum whose epigenetic status has been changed due to exposure of the mother to dioxin will also lead to an altered epigenome in the embryo. The studies of the Seveso accident in Italy documented the illness of the children of the women who conceived as long as 25 years after the dioxin exposure (Baccarelli et al 2008). 

Epigenetic Transgenerational Inheritance

The first animal study to demonstrate transgenerational actions of dioxin was on mouse fertility in 2011 (Bruner-Tran and Osteen, 2011) An important new study, following on this finding, delineates exactly how dioxin can reprogram the epigenome by changing the methylation states of the ova, the sperm and the zygote as found in mice (Manikkan et al 2012). Very low pharmacological doses of TCDD were used, only 0.1% of the oral LD50 so that no direct effects on the mice was seen. This study investigated the F3 generation, which would have had no direct exposure; the F1 and F2 generations could be affected through the germline which can take place in the F0 generation, the generation with direct exposure to the 0.1% oral dose. This study clearly demonstrates the potential of dioxin to promote epigenetic transgenerational inheritance of disease.

Chronic kidney disease in humans is correlated with high dioxin levels (Couture et al 1990). In the mice study, this is the first time a transgenerational kidney histopathology was shown in the unexposed F3 generation of the F0 gestating females exposed to dioxin. The incidence of kidney disease was higher in the F3 generation males. Further the ovarian disease and abnormality included primordial follicle loss and polycystic ovarian disease in the F3 generation. Similar to kidney disease, ovary disease also appears to be the outcome of epigenetic transgenerational inheritance. In some women affected from the Seveso exposure in Italy in 1976, there is a risk of earlier menopause, which reflects a loss of primordial follicle pool (Eskenazi et al 2005). In this study the report of primordial follicle loss and polycystic ovarian disease found in the F3 generation supports previous findings. Pubertal abnormalities were increased only in the female F3 generation animals. The early and delayed onset of puberty are forerunners to different adult health consequences. Early onset puberty results in accelerated bone mineralization and reduced adult height in girls as well as susceptibility to breast tumors. The delayed onset of puberty leads to reduced bone mineralization, emotional stress and metabolic problems (Jacobson-Dickman and Lee 2009). In the current study of rats, the females in the F3 generation showed early onset of puberty while the males showed delayed onset of puberty, indicating sex-specific effects.

These DNA methylation Regions have been located in specific gene coding areas and affect the following biochemical processes in the cell, which means all of these processes will be changed or modified or possibly switched off (Fig. 7). The functions affected by these genes are the cytoskeleton and the Extra Cellular Matrix [ECM or connective tissue]; development; epigenetics, Golgi apparatus, [important for packaging protein for secretion or use in the cell itself]; growth factors; hormones, [beta luteinizing hormone, triggering ovulation in females and production of testosterone in males]; immune response; metabolism and transport across cell wall; proteolysis [breakdown of proteins]; receptors and binding proteins [cholinergic and olfactory receptors]; signaling [response of the cell to signals, such as hormones]; transcription [reading of DNA into messenger RNA, then from mRNA to protein, causing cancers]; translation and protein modification; plus 6 genes that have an unknown function (for full details see Mannikam et al 2012). It is hard to translate all of these modifications into a specific disease but virtually every normal physiological cell process would be affected (Fig. 7).

Thus the former exposure of a gestating female to dioxin promotes an altered fetal gonadal development and epigenetic reprogramming of the male germline that is then transmitted to subsequent generations to contribute to these ovarian diseases to a generation that has never been exposed to these toxic chemicals. The molecular mechanism of epigenetic transgenerational inheritance involves the reprogramming of the germline epigenome during male sex determination. The modified sperm epigenome appears to be permanently reprogrammed similar to an imprinted gene, and is protected from DNA demethylation and reprogramming after fertilization and in the following generations as described above. This allows the epimutations to modify all the somatic cells to promote the disease phenotypes.

Fig. 7 Dioxin promoted F3 generation sperm epimutations. Chromosomal locations for transgenerational differential DNA methylation regions (DMR) (arrowheads). There were 50 DMR in sperm DNA from dioxin lineage rats compared to control lineage rats (Mannikam et al 2012).

This disaster is caused by many environmental chemicals, including vinclozolin, bisphenol-A used in the plastic industry to soften plastics. These results have implications for the human populations that are not only exposed to dioxin but other industrial chemicals yet to be tested. It would begin to explain the global decline in fertility, increases in adult onset disease, the explosion in diabetes Type 2 and global obesity in the developed countries. The terrible tragedy of all this is the potential to transmit them to future generations that have never been exposed.

No one has any idea how long it will take to eradicate these permanent changes in the epigenome or if it is even possible. There is no mention of this present disaster in the popular science magazines as it would necessitate the banning of many chemicals and a total revamp of the plastic industry. 

Error of dioxin is repeated with Bisphenyl A, used in plastics.

All the mistakes we have made with Agent Orange and dioxin are now being repeated with the chemical used to make the clear, hard plastic of polycarbonate, Bisphenol A (BPA). BPA is a diphenol with a similar shape to dioxin, although the halogens (chlorine) have been replaced by hydroxyl (Fig. 9). BPA is also an endocrine disruptor using the same mechanism as dioxin (Manikkam et al 2013). BPA has now been linked to health problems including child development and reproduction, infertility, obesity, hypertension, diabetes, thyroid and central nervous, such as attention deficit disorder (ADHD), as well as cancers such as breast and prostate. The above findings have implications for the human populations that are not only exposed to dioxin but other industrial chemicals, such as DEET (used in insect repellents such as RID), vinclozolin and others, yet to be tested. [Vinclozolin is a common dicarboximide fungicide, used to control diseases, such as blights, rots and molds in vineyards, and on fruits and vegetables such as raspberries, lettuce, kiwi, snap beans, onions and the turf on golf courses.] Vinclozolin is not registered for any use in Australia although DEET is registered and in common use.

Fig. 8 The changes in methylation induced by dioxin and the number
of genes affected in varying cell processes (Manikkam et al 2012)

BPA is used to make certain plastics and epoxy resins. Bisphenol A (BPA) is a synthetic monomer used to manufacture polycarbonate plastics (i.e. food and water containers) and epoxy resins (i.e. canned food linings and it has been in commercial use since 1957. BPA-based plastic is clear and tough, and is used to make a variety of common consumer goods such as baby and water bottles, sports equipment, and CDs and DVDs and for industrial purposes, such as lining water pipes. Water bottles left in the sun, heated in the microwave or in a car can leach out small amounts of BPA. Epoxy resins, containing BPA, are used as coatings on the inside of many food and beverage cans. Cans containing food were lined with a plastic coating to stop the leaching of the soldering metals, used to seal the can. The solution is probably worse. BPA is also used in making thermal paper, such as that used in sales receipts. Since 2008, several governments have questioned its safety, which prompted some retailers to withdraw polycarbonate products. A 2010 report from the United States Food and Drug Administration warned of possible hazards to foetuses, infants, and young children. In September 2010, Canada became the first country to declare BPA a toxic substance. The European Union, Canada, and recently the United States have banned BPA use in baby bottles. 

The 2003-2004 National Health and Nutrition Examination Survey (NHANES III) conducted by the Centers for Disease Control and Prevention (CDC) found detectable levels of BPA in 93% of 2517 urine samples from people six years and older. The CDC NHANES data are considered representative of exposures in the United States. Another reason for concern, especially for parents, may be because some animal studies report effects in foetuses and newborns exposed to BPA.

Food Standards Australia and New Zealand do not consider BPA a health risk {link 01} There are very few reports in the mass media about this problem, whereas it should be front page news, with a campaign to ban its use. Highlighting this problem, instead of pushing Master Chef cooking competitions, would be more useful, but it would necessitate the banning of many chemicals. It would also mean a total reorganization of the production process of the plastic industry and thousands of products, a costly exercise. It also would begin to explain the global decline in fertility, increases in adult onset diseases in children, the explosion in diabetes Type 2, ADHD in children and global obesity in the developed countries. The terrible tragedy of all this is the potential to transmit ill health as a condition of life to future generations that may never have been exposed to these chemicals. No one has any idea how long it will take to eradicate these permanent changes in the epigenome, if these chemicals were to be banned.

Fig. 9 A) Dioxin B) Bisphenol A (BPA) C) DEET (Diethyl toluamide)

AUTISM 

Another factor that is very concerning is the possibility that dioxin contamination of the human diet could be a contributory factor in the development of autism in children. The etiology of autism has been difficult to determine. Autism is multifactorial, some with a genetic basis, either a familial or a spontaneous mutation but others environmental. The Autism Spectrum Disorders (ASD) result from a complex combination of genetic, epigenetic, environmental toxins, air pollution, organophosphates, pesticides and heavy metals. There has been a dramatic increase in ASD since the 1980s. To a certain extent, this is due to both a greater awareness of the disorder but there are likely additional factors contributing to the increase, including environmental factors. The prevalence of ASDs is currently 2 in 100 children in the United States, with numbers increasing each year (Centers for Disease Control and Prevention 2012).There is a difference in sex ratios of sufferers of autism where it is 4 times more common in boys than girls. 

Now there is increasing evidence that autism could be due to changes in the methylation of both DNA in the methylated cytosine base as well as methylation of the histones that control transcription from the DNA. The ASDs may be classed as“epigenopathies, a new term, meaning pathologies resulting from disturbances in the epigenetics of the genome. A number of rare brain disorders have pointed to the disruption of chromatin remodeling and DNA methylation patterns in the developing brain. 

There is substantial evidence from human studies, that human neurons require extensive DNA and histone protein methyl modifications throughout foetal development and post natal life. In the brain, epigenetic programming appears to be most sensitive to environmental and genetic influences in utero and early in postnatal life. Methylation reactions are crucial during this time to develop functional neuron networks. Early modifications in DNA methylation that cause cells to deviate from differentiating into their normal lineage can result in significant decreases or expansions of neuron pools that are irreversible (Zeisel 2011). Methylation patterns also appear to regulate neuron connectivity. Importantly, once adult-like dendritic arbors are stabilized early in postnatal development, large-scale structural plasticity does not appear to be possible (Romand et al. 2011). Prefrontal cortex neurons from subjects with autism show changes in chromatin structures at hundreds of loci genome-wide, revealing considerable overlap between genetic and epigenetic risk maps of developmental brain disorders. (Shulha et al 2012). The post-mortem brain tissue of autistic patients was examined and they identified four significant areas of differentially methylated regions (Ladd Acosta et al, 2013). 

These experiments suggest that epigenetic dysregulation during the period when brain organization develops can lead to a number of neuroanatomical abnormalities, including alterations in brain size and connectivity, that are likely irreversible later in life. (Schaevitz and Sweeney-Berger, 2012). It is suggested that the most critical periods for epigenetic regulation in ASDs occur prenatally, when cells are proliferating actively and differentiating, and very early postnatally, when methylation patterns are necessary to establish normal neuron networks in the brain. 

Exposure to toxins appear to result in lower levels of DNA methylation (Baccarelli and Bollati, 2009). Secondly, there are sex differences in susceptibility to detrimental environmental factors, which would fit with the data on autism. It has also been postulated that low doses of biphenols such as trichlorodiphenyl trichloroethane (DDT) and bisphenol A, may cause incomplete methylation of specific gene regions in the young brain and impair hippocampal neurogenesis across generations. In some circumstances, the epigenetic effects are exerted during in utero-exposure, while in other circumstances, the effects are transmitted across generations via incorporation into the germline cells. For example, exposure to the fungicide vinclozolin early in pregnancy is imprinted in the male lineage, resulting in anxiety behavior and unique patterns of gene expression in relevant brain regions. Similarly, prenatal exposure to polychlorinated dibenzo-p-dioxin (PCDD) and polybrominated diphenyl ethers (PBDE) caused psychomotor deficits in 6-month-old infants and 1- to 6-year-old children, respectively (Herbstman et al, 2010). Since we have shown above that these chemicals, Bisphenyl A and vinclozolin have a similar mechanism of action to dioxin, it is not unreasonable to assume that dioxin could be a contributing environmental factor to the increase in autism. This could occur through exposure of either parent to dioxin even though the children have never been exposed directly to dioxin. This has not been directly examined by epidemiology studies but is an area that needs more research. 

Conclusions 

It is very difficult and expensive to obtain an accurate measure of TCDDs and PCBs in the environment or in animal tissue. Chobtang et al (2011) have highlighted the need for biosensors to detect dioxins in the milk, eggs, fish and the meat food chain. However there is a much cheaper, alternative method, which will give an indication of the severity of a person’s exposure, although not the levels in the environment. This is to measure the levels of the enzyme, cytochrome P450 (CYP1A1) in the blood. This will measure the degree of induction of the enzyme to metabolise dioxin in the liver due to exposure. 

There are more than 13 million deaths every year due to environmental pollutants. Some 24% of diseases are estimated to be caused by environmental exposures that could be averted. In the US, some 148 different environmental chemicals were found in the blood and urine of its population, an indication of the extent of our exposure to environmental chemicals (Hou et al 2012). Rachel Carson, with her book “Silent Spring’’ written in 1962 and strongly refuted by the chemical companies, warned of the real dangers of these very same problems, even though the biological mechanisms were unknown then. The direct effect of TCDDs is not through a genetic mutation effect but because of the ability of the molecule to be taken up in the tissues, diffusion into the cells because of its fat-soluble properties and disruption of the timed transcription of DNA into specific proteins. Once inside the cell it can switch on the ubiquitous transcription factor AhR, which controls almost 2000 different genes, involved in tissue organisation, cell growth and normal cell physiology. This accounts for its huge variable effect on many different tissues. 

More recent publications expand on this method of epigenetic transmission of disease where poor health outcomes in children are associated with harmful maternal exposure to toxins and occupational exposure of the father. This includes congenital abnormalities, obesity, insulin resistance, cardiovascular disease, behavioural disorders and cancer (Soubry et al 2014). The finding that dioxin promotes epigenetic transgenerational inheritance of disease is devastating to those populations directly affected, which includes the entire people of Vietnam, Laos and Cambodia, and all Vietnam Veterans, who fought as allies with the US Army during the Vietnam War from 1961 to 1973. This epigenetic process, by changing the demethylation and remethylation of cytosine bases in the DNA, changes chromatin remodeling, by which genes are selected for transcription by altering the accessibility of gene promoters and regulatory regions to access the genome. 

The molecular mechanism of epigenetic transgenerational inheritance involves the reprogramming of the germline epigenome during male sex determination as well as during female sex determination. The dioxin-modified sperm epigenome appears to be permanently reprogrammed similar to an imprinted gene and is protected from DNA demethylation and reprogramming after fertilization and in the following generations as described above. This allows the epimutations to modify all the somatic cells to promote disease phenotypes. That is why the children of Australian Vietnam veterans who fought in the war are affected, despite the fact the mother would not have been exposed to large doses or even small amounts of dioxin. It is not known how many generations it would take to remove the epimutations, if ever. 

It is now estimated that based on 50 known methylation changes in the rat genome, it would take at least 10 generations or 200 years to restore the human genome to its original epigenetic markers, if only 50 methylation changes occurred and there was no further exposure to dioxin. Currently the world’s female population is facing an increasing incidence of primary ovarian insufficiency, characterised by a loss of follicle reserves in the ovary and an increasing incidence of polycystic ovarian disease, characterised by the presence of anovulatory cysts which could be laid at the feet of these industrial chemicals, still in the environment (Hart et al 2004, Vujovic, 2009). It will need a people`s power revolution to remove these toxins from our environment.

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Footnote:
Link 01 http://www.productsafety.gov.au/content/index.phtml/itemId/971446.