Effects of Dioxins on Human Health : a Review

(TCDD) has been known since 1950s. TCDD is a by-product of herbicide 2,4-dichloroacetophenol (2,4-D) and 2,4,5-trichloroacetophenol (2,4,5-T), but it was first found in fryash of municipal incinerator in 1979 in Japan. In 1998, the survey of municipal incinerators revealed that 105 out of 1,641 produced above the allowed emission level of 80 ng TEQ/m3. Total annual release of dioxins is estimated to be about 5,000 g TEQ in 1997 in Japan. Japanese government started a comprehensive survey for dioxin levels in milk and blood of residents around incinerators, and their health effects. Human effects by dioxin exposures in Western countries were mostly acute and at high level in accidentally and/or occupationally. Health effects of low-dose and long lasting exposure has not been well understood. Certain amount of polychlorinated dibenzo-p-dioxins (PCDD), dibenzofurans (PCDF) and polychlorinated biphenyls (PCB) is accumulated in our body. Mother's milk is also contaminated by PCDD/PCDF. Health effects of the polychlorinated chemicals are summarized, and the necessity of regulations and recommendations for making a guideline is discussed in this review. Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF) occur as unwanted by-products of thermal processes, so the major part of their environmental distribution is caused by human activity1). Burning waste materials that may contain chlorine, such as plastics, pesticide-treated wastes, wood treated with pentachlorophenol (PCP), other polychlori-nated chemicals can produce PCDD/PCDF1). Waste incineration is the most important sources of emission now, although herbicide industry, chlorine and paper industry, melting processes, car traffic, etc. yield PCDD/PCDF. Sakurai et al2) studied on the origin of dioxin pollution from the isomer pattern in Kasumigaura, Ibaragi, Japan, and estimated that it was attributable to air (39%), CNP (22%), and PCP (22%). PCDDs have also been detected in cigarette smoke. Side stream smoke contained more PCDD/PCDF (1.3-9.2 pg-TEQ/pack)(Shiosaki et al, personal communication). The Environmental Agency estimated annual release of dioxins in Japan being about 5,000 g3) (Table 1). Japan becomes one of the most heavily polluted Table 1. Estimated amount of dioxins in Japan.

country by dioxins in the world.
The US Environmental Protection Agency (EPA) has regulated dioxin (2,3,7,8-TCDD) as a carcinogen based on the positive animal data and the compatible epidemiological findings of occupational exposure1).2,3,7,8-TCDD is classified to group 1 carcinogen at the IARC review work in February 19974).PCDD and PCDF have many isomers.Of the 75 PCDD isomers and 135 PCDF isomers, only 7 and 10, respectively, are likely to have toxic properties similar to 2,3,7,8-TCDD.Thirteen polychlorinated biphenyl (PCB) are also have toxicity among 206 different PCB congeners.The toxicity are summed based upon their toxicity equivalency factor (TEF)(Figure 1.).Determination of TEF still contained some problem, but, so far, it is practical for evaluating risk for public health.Biological effects other than cancer caused by a background level exposure is unclear.Lowered immune function and possible activity as endocrine disrupter are plausible human toxicity from animal experimentsl).It works at very low concentration at level of ng/kg (pg/g) body weight.The determination of human burden (cumulative dose) in such a low concentration had been impossible until the high resolution mass-spectrometry have been developed for quantitative measurement.
WHO and several countries develop regulations and recommendations to protect public health.Regulations should be enforced by the law.This review deals with recent situation of dioxin research in Japan and world, mainly focused on human health effects.

PRODUCTION OF DIOXINS AND RELATED SUBSTANCE
Dioxins are produced from chlorinated materials and aromatic carbohydrates by burning trash in an incinerator.When there is catalysts, like iron, the producibility becomes high.When two oxygens combine two benzenes (or chlorinated benzene), it becomes para-dibenzodioxins, and when one oxygen combines two benzenes, it is called dibenzofuran.Both chemicals could have up to 8 chlorines, so the number of isomers is many; 75 for PCDDs, and 135 for PCDFs.All these compounds are called congeners.Some PCBs (polychlorinated biphenyls) have similar character with dioxins, because of their flat structure.These four isomers among 206 isomers are called coplanar PCBs.Toxicity of these congeners is evaluated based on the 2,3,7,8-TCDD, and expressed as toxic equivalency factor (TEF).Presence of four chlorine atoms at positions 2,3,7, and 8 on the dioxin molecules seems to cause toxicity (Figure 1.).The toxicity are summed up based upon their toxicity equivalents concentration (TEQ), and expressed as pg TEQ/g lipid (ppt).
A general observation for human background contamination   is that OCDD is the most abundant isomer, followed by the 2,3,7,8-substituted hepta-and hexa-chloro-congeners.2,3,7,8-TCDD is normally less abundant than PeCDD.The toxicity is considered to be additive.The determination of TEF is based upon the activation of Ah receptor, induction of CYP1A1, in animal experiment, for chemicals with dioxinlike structure (Table 2).So the other effects that are not via Ah receptor are not evaluated by TEQ expression.The oxygen bond between two benzenes could be substituted to sulfur or azide (Figure 2.).Similarly, chlorine could be replaced by bromium.Toxicity of bromodioxins is considered to be similar to that of chlorinated dioxins5,6).Recent increase of polybromobiphenyl (PBB) calls attention by environmentalists, whether or not the chemicals have human health effects.
PBB is now widely used for inflammable clothes.More sophisticated experimental design is necessary to screen toxicological effects7).Until 1970, PCDD/PCDF had been made as a contaminant in the manufacturing process of certain chlorinated organic chemicals, such as 2,4-D and 2,4,5-T.2,4,5-T has been widely sprayed for weed control on lands, and along roadways throughout the world.2,4,5-T was a component of Agent Orange, which was heavily used by the US military in the Vietnam War8,9).Currently emission from incinerator sources becomes problem.Management practices and applied technologies influenced the emission of dioxins10-12).Recent report form Osaka showed extremely high concentration of PCDD/PCDF around incinerator due to smoke washing system (Watanabe et al. in preparation).

UPTAKE BY HUMANS AND ACCUMULATION
Three routes are considered for human exposure; eating food, breathing polluted air, and skin contact with contaminated soil and materials13-16).
Plants take up only very small amounts of PCDDs by their roots.However, spinach and several vegetables contained dioxins at certain level17).Most of the PCDDs found on the Plants and animals can condense through respiration or food chains.We are exposed to 0.52-4.52pg TEQ/kg/day in urban area, 0.5-3.5 pg TEQ/kg/day in towns, and 0.29-3.29 pg TEQ/kg/day in background area3)(Table 3).The actual intake of PCDDs from food for any one person will depend on the amount and type of food consumed and the level of contamination 13).Japanese takes a lot of fishes, so the frequent fish eaters consume more PCDD/PCDF/PCB.Coast-side fishes, especially from areas contaminated with chemicals, contained more dioxins17).Dioxin congeners are known to be accumulated mostly in fat and liver of fishes and animals.
In Japan, 90% of the daily intake of PCDDs, PCDFs, and other dioxin-like compounds is estimated to come from food, primarily fish and meat in general population.Japanese levels of PCDD/PCDF in blood from the general population ranged from 0.13 to 29 pg TEQ/g lipid (8.21 pg TEQ/g lipid in average) in Saitama prefecture.The concentrations in humans are higher in industrialized countries, being about 15 pg TEQ/g lipid and normally below 10 pg TEQ/g lipid.These values are several orders of magnitude lower than those observed in accidentally or occupationally exposed individuals 4) .Dioxins bind Ah receptor in cytoplasm, and the complex moves into the nucleus, where it binds to Arnt to bind gene activator sites.CYP (p450) 1AI is a representative product, but many other products would be yielded.latency periods suggest its promoter activity (Table 5).
Spindler37) evaluated the carcinogenic risk of soot on basis of the unit risk concept for relative carcinogenic risk of dioxins compared to polyaromatic hydrocarbons (PAH).PAHs, like benzo(a)pyrene, dibenzo(a)anthracene, etc., are much more important by factors of 25 to 500 in comparison to dioxins.
Follow-up study of the Ranch Hand cohort showed the increased mean diastolic blood pressure in those with current serum lipid TCDD levels from 15 to 33.3 pg/g lipid9).The RR of mortality from heart and circulatory diseases increased to 1.96 (95%CI; 1.15-3.34)and 2.48 (95%CI; 1.32-4.66),respectively, in BASF workers with serum TCDD lipid levels of >348 pg/g.

IMMUNE FUNCTION
Many animal studies have shown adverse effects of PCDD/PCDF and PCBs on the immune system.The most consistent finding is thymic atrophy.In utero and lactational exposure is a more sensitive period for the immunotoxic effects than adult exposure39).In vitro studies of human venous blood and lymphocyte fractions incubated with low doses of TCDDs, demonstrated a decrease in B-cells and CD4+ (helper) T-cells and in relative increase in CD8+(suppressor) T cells40).In human studies, there is no consistent exposure-related changes in lymphocyte subset and stimulation.Nagayama et al41)reported that the increased T4/T8 ratio according to the plasma dioxin level.
The changes in the T-lymphocyte population could persist into later child-or adulthood and could result in immune suppression, allergy or autoimmunity.
Zober et al31) found a significant increase in the incidence of infectious and parasitic diseases in TCDD exposed workers in the 35 year period after the BASF accident.A two fold increase in the incidence of upper respiratory tract infection and higher incidence of appendicitis were reported.Respiratory involvement was also present in Yusho patients42).Although increased WBC counts was reported in Missouri study28), a follow-up study of the same population found no difference in WBC, RBC and platelet count between exposure and non-exposure.

ENDOCRINE DISRUPTER
Several organochlorines have been identified as endocrine disrupting agents43,44).o,p'-DDT and p,p'-DDE act directly through binding to steroid hormone receptors .Other dioxin-like compounds operate through a variety of mechanisms, one of them being the alteration of hormone metabolism through induction of cytochrome CYP dependent enzymes47-49).
The effects of TCDD exposure on gonadal function has not been well studied.A health study of US veterans showed a significant association between testicular size and serum TCDD levels, although no alterations in sperm count or the percentage of abnormal sperm was observed9).In workers at two 2,4,5-T factories, serum TCDD levels were positively correlated with follicle stimulating hormone (>140 pg-TEQ/g lipid) and luteinizing hormone levels (>1,860 pg-TEQ/g lipid) and inversely correlated with total testosterone levels (>140 pg TEQ/g lipid)50).
Pregnancy outcomes seems to show no significant alterations in the incidence of spontaneous abortions among Vietnam War ranchers.An increased incidence of spontaneous abortion was reported in women living close to a herbicide manufacturing factory in Sweden51).The residents were exposed to phenoxy acids, chlorophenols, TCDD and PCDF which were released into the soil and groundwater.The sex ratio is another interesting point, because female/male ratio in Seveso between 1977 and 1984 was 48/26, but it became 64/60 between 1985 and 199452).Thus, the results of the human reproductive toxicity studies are inconclusive.There are some other hormonal effects.A 35-year follow-up study of TCDD exposed workers at BASF accident found a significant increase in the incidence of thyroid disease, as compared to an age-matched referent group21).A strong positive association was found between glucose intolerance or increased risk of diabetes mellitus and TCDD serum levels9).Interference of TCDD with secretion or synthesis of bioactive ACTH is considered to affect adrenal steroidogenesis by animal experiment53).

DEVELOPMENT
Small amounts of PCDD/Fs pass the placental barrier during pregnancy.The embryo and fetus are exposed during a critical period of organ growth and development76).After birth the breast-fed infant is exposed to relatively high levels of these contaminants.
The overall risk of having a child with birth defects was not significantly increased in the Vietnam veterans (OR of 0.97, 95% CI=0.83-1.14),but some birth defects, such as spina bifida, cleft lips, and congenital tumors (dermoid cysts, teratomas, hepatoblastomas, central nervous system tumors, and Wilms tumors), were present at higher proportion among veterans father.Malformations of nervous system, cardiovascular system, genital system and urinary system may be caused by father's exposure to TCDD.Koopman-Esseboom et al54) reported relationship between maternal and cord blood plasma level of dioxins in 418 mother-infant pairs and neurological and psychomoter development.They recognized a small delay in psychomotor development, and alterations in the thyroid hormone status.
Ilsen et al55) observed signs of enhanced neurological maturation in children at 2 years and 7 months in relation to the perinatal load of dioxins (19 low exposure group; 8.7-28.0pg TEQ/g milkfat, 19 high exposure group; 29.2-62.7 pg TEQ/g milkfat.)Although all psychomotor, neurological and laboratory findings were within the normal range, the signs of higher reflexes and enhanced neuromoter maturation should be regarded as a warning sign.These findings may be due to thyroxine agonistic action of dioxins43,44).Nagayama et al41) reported lowered plasma T4 levels in breast fed babies.
Newborns from Yu-Cheng mothers who were exposed in utero to both PCBs and PCDFs were smaller at birth than newborns from unexposed mothers.Correlation analysis revealed a statistically significant inverse relationship between aryl hydrocarbon hydroxylase (AHH) activity and weight of the newborb56).

OTHER EFFECTS
Chloracne has been a well known skin lesion and often used as a surrogate marker of dioxin exposure57-60).Chloracne is characterized by follicular hyperkeratosis (comedones) occurring with or without cysts and pustules61,62).Unlike adolescent acne, chloracne may involve almost every follicle in an involved area, such as face, neck, upper arms, back, chest, abdomen, outer thighs, and genitalia.In mild cases, the lesion may clear several months after exposure ceases, but they may persist 30 years in severe cases60, 63,64).Porphyria cutanea tarda, hypertrichosis, hirsutism, and hyperpigmentation were also reported in TCDD-exposed workers.In case Kanemi Yusho , many patients are still suffering from various skin lesions 30 years later64).
In Seveso, erythema and edema of exposed areas, vesiculobollus and necrotic lesions and papulonodular lesions were found in 447 people 20-40 days after the accident, and 34 of these people later developed chloroacne65).Chloracone level in Seveso was 1,770-10,400 pg TEQ/g lipid, in Missouri 577-2,310 pg TEQ/g lipid, and in Operational Ranch Hand veterans 618-4,477 pg TEQ/g lipid.In Seveso children, 12,100-56,000 pg TEQ/g lipid blood level was associated with type 4 chloracne, and 823-7,420 pg TEQ/g lipid with type 3 chloracne.Chloracne is a good marker of dioxin exposure, but its absence does not preclude such exposure.Schuster et al66) estimated that mean body burden of workers with chloracne was 3,200 ng versus 72 ng for general population.
Hepatic toxicity was observed in acute and subacute exposure of TCDD.Hypercholestelonemia, hyperlipemia, and hyperphospholipidemia are observed in about half patients with chloracne.At 10 years post exposure, most of the biochemical changes were not detected except for high cholesterol level67).A slight increase in GGT and ALT occurred in the highest exposure group in Seveso residents, but returned to baseline levels within 3 years of the initial exposure68).A positive trend for GGT and alkaline phosphatase was observed in Missouri residents living in a TCDD-contaminated area.
Uroporphyria or urinary porphyrins did not indicate any definitive changes in the residents exposed for up to 11 years.In general, the effect is mild and transient.Kidney lesions have not been reported in any of the several studies on occupational exposure or in the cohort from Seveso.
Neurological effects also appear by dioxin exposure.Subjective signs, such as lassitude, weakness of the lower limbs, muscular pains, sleepiness or sleeplessness, increased perspiration, loss of appetite, headaches, and mental and sexual disorders were reported in several workers with severe chloracne who had been exposed to TCDD69,70).Neurological symptoms persisted in these workers for up to 10 years.In German accident, peripheral neuropathy, sensory impairment , tendency to orthostatic collapse, and reading difficulties were reported71) .Depression, hypochondria, hysteria, and schizophrenia were found more often in Vietnam veterans exposed to herbicides than in the control groups of veterans29).The blood concentration of dioxins was 16.1-80.4pg TEQ/g lipid with a mean of 31 pg TEQ/g lipid in Peper's study in Germany71).Gastrointestinal effects, such as peptic ulcer, was once reported, but a study of Vietnam veterans failed to find such effects .Occupational cohort study by Calvert et al .72)did not find any gastrointestinal disease difference between cases (220 pg TEQ/g lipid) and controls (7 pg TEQ/g lipid) .In Seveso residents, increased risk of gastric cancer was reported once35) .LEGISLATION WHO and several countries develop regulations and recommendations to protect public health .Regulations can be enforced by law (Table 6).Environmental Agency of Japan set the level of dioxin emission to be less than 80 ng TEQ/m3 , in which situation human exposure would be less than 10 pg TEQ/kg/day.WHO, Japan and several other countries have used a TDI approach, based on the two-year Kociba's experi-ment73), which focused on the liver tumors in female Sprague-Dawley rats.It resulted in an upper bound estimate of an excess of one in a million cancer risk from exposure to 6 fg/kg/day.They suggested that 1 ng/kg/day was a NOAEL for carcinogenesis.Applying a 10-fold safety factor for interspecies extrapolation and a 10-fold "correction" for rat/human pharmacokinetic differences, resulted in a TDI of 10 ng/kg/day.The WHO also suggested that a TDI would be appropriate based on their estimation of a NOAEL of 1 ng/kg/day bases on the Murray54) multigeneration reproduction study.WHO's TDI is proposed to change to 1-4 pg TEQ/kg/day in 1998 considering risk of lowered spermatogenesis or risk of endometriosis.
The linearized multi-stage (LMS) model was used as a The toxicity equivalent (TEQ) of TCDD is calculated by multiplying the exposure level of a particular dioxin-like compound by its toxicity equivalency factor (TEF).Given that current exposure to total TCDD equivalents is approximately 1-3 pg/kg/day, EPA deemed it inappropriate to establish RID which would likely be less than the current average daily intake.FDA recommends against consuming fish and shellfish with TCDD levels greater than 50 pg TEQ/g.Such levels have resulted in the closing of several commercial fishing areas.EPA advises that children should not have more than 1 ng/L water in one day, or more than 10 pg/L per day for long-term exposure.For adult, it should be less than 40 pg/L in drinking water.
In Japan, the concentration of dioxins in the air around exhausted smoke from the chimney is restricted under 0.8 pg/m3.Soil levels for legislation in Germany and Italy is shown in Table 6.Strict legislation should be effective to decrease background level of dioxins in the body74,75).Such legislation has not yet determined in Japan.First removal of contaminated soil (more than 8,000 pg/g dry weight) was recently performed around the municipal incinerator in Osaka.Agency for Toxic Substances and Disease Registry in U.S.A. proposed a policy guideline78,79).They proposed decision framework for sites contaminated with dioxin and dioxin like compounds (

Figure 2 .
Figure 2. Dioxins and related compounds.Various compounds which are considered to show similar effects of PCDDs/PCDFs.

Table 2 .
The TEQ concept by European Center of Environmental Health of WHO and International Program on Chemical Safety.

Table 3 .
Estimated intake of dioxins by average Japanese.

Table 4 .
Biological half-life (year) of dioxins in human body.

Table 6 .
Legislation of dioxins in Germany and Italy.
* Residents may stay in polluted area , but pregnant women and children should stay outside during daytime.Table7.ATSDR's decision framework for sites contaminated with dioxin and dioxin-like compounds in U.S.A.

Table 8 .
Research Program in Japan.EPA's default position.Application of this model also to the bioassay results from Kociba73).Approaches for extrapolation beyond the range of observation are being explored.The EPA has not regulated dioxin based on its non-cancer effects, although believing that the use of the LMS model for carcinogenesis would be protective for non-cancer effects as well.

Table 7
Ministry of Health and Welfare, Environmental Agency and local government.Current programs are shown in Table8.CONCLUSIONData from Europe suggest that dioxin levels in human tissues may have decreased rapidly over the past 4-6 years.Environmental sources, regulations, and enforcement may vary between the United States and Germany, so a clear decrease was shown in levels from the 1980's and 1990's only in Germany74).Annual release of dioxins in Germany now is only 40 g.It is more than 100 times less than that of Japan.Urgent survey of the municipal incinerators revealed nearly 1% incinerators emit dioxins above allowable emission level.Health check-up of residents around such incinerators including control is carried out in 6 different prefectures, so the result should be shown in the near future.Holistic environmental control system in these areas and follow-up system for the residents are necessary in Japan.Monographs on the Evaluation of Carcinogenic Risks to Humans.Vol 69.Polychlorinated dibenzo-paradiozins and polychlorinated dibenzofurans.IARC , Lyon, 1998. 5. Schulz-Schalge T, Koch E, Schwind H-K , Hutchinger Ott, Neubert D. Comparative study on the inductive potency of TCDD and TBrDD with three 2,3,7,8-mixedhalogenated dioxins in liver microsomes of male rats.