DETERMINANTS OF SENSITIVITY TO DIOXIN-INDUCED HEALTH EFFECTS

Project leader: Matti Viluksela, National Public Health Institute (KTL), Laboratory of Toxicology, P.O. Box 95, FIN-70701 Kuopio, Finland, tel. +358-17-201329, e-mail: Matti.Viluksela@ktl.fi
 
 

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TIIVISTELMÄ SUOMEKSI

Researchers:
National Public Health Institute, Laboratory of Toxicology:
Jouni T. Tuomisto, tel.+358-17-201305, e-mail: Jouni.Tuomisto@ktl.fi
Ulla Simanainen, tel.+358-17-201177, e-mail: Ulla.Simanainen@ktl.fi
Hanna Kattainen, tel.+358-17-201177, e-mail: Hanna.Kattainen@ktl.fi

Consortium: Environmental health risks of dioxin
Financing SYTTY organisation: The Academy of Finland
Funding from SYTTY / Total funding of project (€): 159122/335849
Person-months of work funded by SYTTY / Total person-months of work: 63/127

KEY WORDS: AH receptor, dioxins, TCDD, tooth, bone, reproduction
 

EXTENDED ABSTRACT

1 Introduction

As persistent and highly toxic environmental contaminants with largely obscure mechanisms of action, polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) are a potentially significant environmental health problem, which is not merely a remote theoretical possibility. For example, recent epidemiological findings suggest that current higher European background exposure via mother’s milk may result in mineralization defects of developing teeth in children (Alaluusua et al., Lancet, 353:206, 1999). Gaps of critical information are reflected as uncertainty in the current assessment of health risks of PCDD/Fs. The main difficulties of the human risk assessment of PCDD/Fs are: (1) The mechanisms of toxicity are not adequately known, (2) Huge species and strain differences in sensitivity to some, but not all endpoints of toxicity complicate the species-to-species extrapolation, and (3) The most relevant and sensitive endpoint for human is not known.

An animal model suitable for studying mechanisms of dioxin toxicity has been established in our laboratory (Pohjanvirta & Tuomisto, Pharmacol. Rev. 46:483-549, 1994). This model is based on exceptionally wide sensitivity difference between two rat strains, the sensitive Long-Evans (Turku/AB; L-E) rats and the resistant Han/Wistar (Kuopio; H/W) rats, which differ by >1000-fold in terms of acute toxicity of TCDD. However, the sensitivity difference is endpoint dependent, as it is only about 100-fold for tumor promotion (Viluksela et al., Cancer Res. 69:6911-6920, 2000) or almost missing for embryotoxicity and thymus atrophy. The aryl hydrocarbon receptor (AHR) of H/W rats was recently shown to harbor a point mutation, which results in an altered transactivation domain and a smaller receptor protein (Pohjanvirta et al., Mol. Pharmacol. 54:86-93, 1998). Two different genes mediate the resistance of H/W rats to TCDD. Isolation of these genes into different rat lines (Tuomisto et al., 1999) made it possible to study the roles and significance of these alleles in dioxin-induced health effects. In this project, the new rat lines (as well as H/W and L-E rats) were used as tools to identify and characterize factors that determine sensitivity to PCDD/F-induced health effects. The studies were focused on clinically relevant and sensitive endpoints, such as dental defects and male reproduction disorders, and the ultimate goal is to improve the risk assessment of PCDD/Fs.

2 Methods

H/W and L-E rats as well as the H/W*L-E -derived new rat lines A, B and C (see below) were bred and maintained under specific pathogen free conditions at the National Public Health Institute (Kuopio, Finland). Depending on the study, adult, neonatal or pregnant animals were exposed to different dose-levels of PCDDs. A variety of endpoints of toxicity were examined using appropriate methodology.

3 Results and Discussion

The roles of the resistance genes in sensitivity to dioxin toxicity
The dioxin resistance genes of the resistant H/W rats were segregated to new rat lines using genetic crossing together with selection based on sensitivity to TCDD and the AHR phenotype (Tuomisto et al., 1999). Line A has the mutated H/W type AHR (allele Ahrhw/hw), line B has an allele Bhw/hw of a currently unidentified gene B, and line C neither of these resistance alleles. Lines A, B and C exhibit highly different LD50 values for TCDD: >10000, 830 and 40 µg/kg in males, respectively, and >2 000, 410 and 19 µg/kg in females, respectively. Dose-response analysis on short-term effects of PCDDs indicated that the AHR-mediated effects can be classified into two different categories based on endpoint-dependent differential efficacy (magnitude of effect) in A and C lines, as well as in H/W and L-E strains (Simanainen et al., 2002a, b). Effects that are similar in these rat lines or strains, and hence independent of the genotypical variation of AHR, are called type I endpoints. These endpoints include cytochrome P450 (CYP) 1A induction and thymus atrophy. Type II endpoints, on the other hand, show suppressed efficacy (or potency) in rats with Ahrhw/hw genotype (H/W and line A rats), and these endpoints include body weight loss, liver toxicity, increased serum bilirubin and free fatty acid levels as well as decreased serum thyroxine levels. Efficacy ratio of 0.5 between resistant and sensitive rat strains/lines was selected as the classification criterion. The decreased ability of the Ahrhw/hw genotype to mediate dioxin toxicity can be localized to steps beyond DNA-binding, i.e. the AHR-dependent gene expression.

Examination of line A, B and C offspring exposed to TCDD in utero / lactationally confirmed earlier findings about high sensitivity of developing male reproductive system (Simanainen et al., 2000), and discovered two new highly sensitive endpoints of dioxin toxicity: the tooth and the bone development (Kattainen et al., 2001; 2002; in preparation). In general, the lines showed differences in their sensitivity to these endpoints, but the differences were smaller than previously shown for lethality (see below).

In spite of their enormous sensitivity difference to lethality of TCDD as adults, H/W and L-E rats did not differ markedly in their sensitivity to embryolethality (Huuskonen et al., Toxicol. Appl. Pharmacol. 124:174-180, 1994). Our experiments on neonatal A and B rats indicated that the resistance of line A rats to a huge dose of TCDD (1000 µg/kg) develops between postnatal days (PND) 2-5 (Tuomisto et al., in preparation). On the contrary, the resistance of B rats develops much later, between PND 14-28. Therefore, the Ahrhw/hw allele accounts for the high resistance to lethality already during early neonatal life, but the Bhw/hw allele results in moderate resistance that develops by weaning.

Effects of dioxins on teeth
Effects of low dose in utero / lactational TCDD exposure on tooth development was studied in line A, B and C rats after a single oral dose of TCDD on gestational day 15 (Kattainen et al., 2001). TCDD at 1 µg/kg completely arrested the development of the lower third molars in 50 and 60% of line C females and males, respectively, and in 5-6% of line A and B females. TCDD also dose-dependently delayed the eruption and diminished the size of the third molars. Mesio-distal length of the third molars was significantly decreased already at the lowest dose-level (0.03 µg/kg) in lines A and C and at 0.1 µg/kg in line B. Therefore, perinatal tooth development is one of the most sensitive endpoints of TCDD-induced toxicity. Toxicokinetic data indicate that the maternal lowest observable adverse effect level (LOAEL) 0.03 µg/kg is likely to result in a maternal lipid based tissue concentration that is very similar with the mother’s milk PCDD/F concentrations associated with molar mineralization defects (Alaluusua et al., Lancet, 353:206, 1999). The resistance alleles Ahrhw/hw and Bhw/hw seem to decrease the sensitivity to arrested third molar development, but they had no influence on the decrease in mesio-distal length. Time-course studies indicated that the critical window of sensitivity for the third molar development is during early morphogenesis, from tooth initiation (GD20) to the early bud stage (PND0), and that the primary target is the dental epithelium (Kattainen et al., 2002).

Exposure of adult rats to TCDD did not affect molars, but caused pulpal perforations, enamel discoloration and impaired dentin formation in continuously erupting incisors at high dose-levels (Kiukkonen et al., 2002). The defects included odontoblast and pulpal cell death, as well as squamous metaplasia and pronounced proliferation of the postsecretory enamel organ. H/W and L-E rats were similar in their sensitivity, which indicates that this is a type I effect.

Effects of dioxins on bones
Long bones of rats exposed to TCDD as adults were analyzed using peripheral quantitative computed tomography (pQCT), three-point bending test and histomorphometry (Jämsä et al., 2001). TCDD treatment was shown for the first time to impair bone quality and modeling. The changes included dose-dependent decreases in mechanical strength as well as altered geometry and bone mineral density. LOAEL for bone effects was 1.7 µg/kg, and L-E rats were more sensitive than H/W rats (type II effect). After in utero / lactational exposure the changes in offspring were observed at lower doses than after adult exposure, indicating that bone development is a sensitive endpoint (Kattainen et al., in preparation).

Effects of dioxins on male reproduction system
A stereological analysis on testes of H/W and L-E rats exposed to TCDD as adults indicated that H/W rats are completely resistant to testicular toxicity of TCDD at dose-levels as high as 1000 µg/kg (Simanainen et al., in preparation). In L-E rats the amount of Sertoli cells and all spermatogenic cell types were decreased after a single dose of 20 µg/kg TCDD. Decreases were observed also in serum testosterone, estradiol and LH concentrations. Leydig cells were not affected. Also adult line C rats were clearly more sensitive to the male reproduction endpoints than line A and B rats (type II effect). The changes were limited to overtly toxic dose-levels.

Effects of in utero / lactational exposure of A, B and C line rats to low doses of TCDD on male reproduction parameters were assessed in the offspring at the age of 70 days (Simanainen et al., 2000; in preparation). Epididymal sperm counts decreased significantly in line C and B rats at maternal dose level of 0.3 and 1 µg/kg, respectively, and daily sperm production at 0.1 and 1 µg/kg and above, respectively. Weights of ventral prostate and cauda epididymis were decreased in lines C and B only at 1 µg/kg. Thus, the rank order of sensitivity among the lines is C>B>A

Accumulation of biliverdin and hepatic peliosis – a new type of dioxin-induced liver toxicity
During cross-breeding of H/W and L-E rats a new type of liver toxicity characterized by dark green or black pigment and swollen livers was found (Niittynen et al., 2002). This “black liver syndrome” was seen most frequently in intermediately resistant rats with at least one resistance allele (Ahrhw or Bhw) after a large dose (>300 µg/kg) of TCDD and a follow-up period more than three weeks. It is thus a type II dioxin effect. Bilirubin metabolism was studied as a potential source of the pigment. The main source of bilirubin is the heme from degrading erythrocytes. TCDD had no influence on the life span of erythrocytes in C rats, which also show large increases in serum bilirubin. The pigment fractions were separated by thin layer chromatography and then analyzed by HPLC and electrospray mass spectrometry. The pigment was found out to consist of biliverdin and several biliverdin-related compounds. In liver histopathology, progressive sinusoidal distension and hepatic peliosis with membrane-bound cysts were seen. The primary biochemical alteration leading to this syndrome remains to be elucidated.

Characterization of abnormal sensitivity of H/W rats to hexaCDD
Structure-activity correlation studies were carried out to characterize the abnormal sensitivity of H/W rats to higher chlorinated dioxins, especially to hexaCDD (Viluksela et al., 1998; Simanainen et al., 2002a). In general, TCDD is by far the most toxic dioxin congener, and the toxicity (in terms of all endpoints) decreases with increasing chlorination (TCDD > pentaCDD > hexaCDD > heptaCDD). This fact is utilized in the toxic equivalency factor (TEF) concept, which forms the basis of the current risk assessment of halogenated dioxins and related compounds. In H/W rats, however, the rank order of potency for mortality is hexaCDD > heptaCDD > pentaCDD > TCDD. Our studies showed that unlike mortality, all other endpoints studied followed the normal rank order of potency. Our results are consistent with the current TEF concept, and indicate that TEFs derived from mortality data are not necessarily valid for non-lethal endpoints. The survival time, body weight loss, and necropsy findings were different in rats given hexaCDD than in rats given other dioxin congeners. This suggests a different and probably Ahrhw/hw independent lethal mechanism of hexaCDD in H/W rats.

4 Conclusions

1. The exceptional resistance of H/W rats to TCDD lethality is determined by two alleles, the deviant AHR allele Ahrhw/hw and the allele Bhw/hw of a currently unidentified gene B. The Ahrhw/hw allele is the major determinant of the resistance, and it is effective already during early neonatal life. Moderate resistance resulting from Bhw/hw develops by weaning.
2. There are two different AHR-mediated effects of dioxins leading to type I and type II endpoints. Type I endpoints (CYP1A induction, thymus atrophy, incisor defects) show similar sensitivity in resistant rats with Ahrhw/hw and in sensitive rats with wild type AHR. Type II endpoints (body weight loss, liver toxicity, biliverdin accumulation, increased serum bilirubin and free fatty acid levels, decreased serum thyroxine levels, male reproduction toxicity, arrested molar development) show lower efficacy in rats with Ahrhw/hw. The mechanistic difference seems to localize in the AHR transactivation domain.
3. In addition to developmental male reproduction toxicity, tooth development and bone development are among the most sensitive endpoints of dioxin toxicity. The resistance alleles Ahrhw/hw and Bhw/hw are linked to increased resistance to most parameters analyzed.
4. TCDD induces two types of dental defects in rats. First, the early morphogenesis of molars involving epithelial-mesenchymal interactions is sensitive to very low doses of TCDD and responds by an arrest of early tooth development. The major target is the dental epithelium. Toxicological significance of these defects is also emphasized by the developmental similarity of human teeth with rat molars, and the results strengthen the biological plausibility of the PCDD/F-associated molar defects reported previously in children. Second, the matrix formation of continuously erupting rat incisors is impaired at high doses of TCDD due to defects in secretory/postsecretory cells. The major targets in incisors are the ectomesenchymal odontoblasts and the epithelial enamel organ.
5. Exposure of adult or prenatal/neonatal rats to TCDD causes decreased biomechanical strength, altered geometry and mineral density of long bones, probably as a result of impaired bone modeling.
6. The resistance alleles Ahrhw/hw and Bhw/hw are associated with high and moderate resistance of developing and adult male reproductive system to TCDD, respectively.
7. TCDD causes a new type of liver toxicity, the black liver syndrome, characterized by biliverdin accumulation and hepatic peliosis in intermediately resistant rats. Increase in serum bilirubin levels in C rats is not caused by shortened life span of erythrocytes.
8. H/W rats are abnormally sensitive to lethality of hexaCDD compared to other dioxin congeners, but not to the other endpoints of toxicity. This may be due to a different mechanism causing lethality.
 
 

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