Effect of Grandmaternal Smoking on Body Size and Proportions at Birth.

Many long-term adverse effects of smoking during pregnancy are known. Increasingly, adverse effects in the grandchild after grandmaternal smoking during pregnancy are reported. We explored this in a birth cohort of 24,000 grandmother-mother-child triads identified from the Finnish Medical Birth Register in 1991-2016. Multiple logistic regression was used to analyze the association between any smoking during pregnancy by both grandmother and mother, or only grandmother or mother on adverse birth outcomes. No smoking by neither grandmother nor mother was used as the reference. As endpoints, preterm birth, low birth weight, small for gestational age (birth weight, birth length, head circumference), and body proportionality (low ponderal index, high brain-to-body ratio, high head-to-length ratio) were included. Smoking by both grandmother and mother was consistently associated with higher risks than smoking only by the mother. Birth length and weight were especially sensitive to (grand)maternal smoking. In conclusion, the combined effect of grandmaternal and maternal smoking is associated with higher risks than only maternal smoking.

Endocrine, metabolic and apical effects of in utero and lactational exposure to non-dioxin-like 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB 180): A postnatal follow-up study in rats.

PCB 180 is a persistent and abundant non-dioxin-like PCB (NDL-PCB). We determined the developmental toxicity profile of ultrapure PCB 180 in developing offspring following in utero and lactational exposure with the focus on endocrine, metabolic and retinoid system alterations. Pregnant rats were given total doses of 0, 10, 30, 100, 300 or 1000 mg PCB 180/kg bw on gestational days 7-10 by oral gavage, and the offspring were sampled on postnatal days (PND) 7, 35 and 84. Decreased serum testosterone and triiodothyronine concentrations on PND 84, altered liver retinoid levels, increased liver weights and induced 7-pentoxyresorufin O-dealkylase (PROD) activity were the sensitive effects used for margin of exposure (MoE) calculations. Liver weights were increased together with induction of the metabolizing enzymes cytochrome P450 (CYP) 2B1, CYP3A1, and CYP1A1. Less sensitive effects included decreased serum estradiol and increased luteinizing hormone levels in females, decreased prostate and seminal vesicle weight and increased pituitary weight in males, increased cortical bone area and thickness of tibial diaphysis in females and decreased cortical bone mineral density in males. Developmental toxicity profiles were partly different in male and female offspring, males being more sensitive to increased liver weight, PROD induction and decreased thyroxine concentrations. MoE assessment indicated that the 95th percentile of current maternal PCB 180 concentrations do not exceed the estimated tolerable human lipid-based PCB 180 concentration. Although PCB 180 is much less potent than dioxin-like compounds, it shares several toxicological targets suggesting a potential for interactions.

Toxicity of colloidal silver products and their marketing claims in Finland.

AIMS: The aim was to investigate the marketing practices, beliefs and health claims regarding the use of colloidal silver in Finland. Silver nanoparticles (AgNPs) are potentially toxic due to their small size and Ag+-release capabilities, and the use of colloidal silver products containing AgNPs can cause a wide variety of adverse effects such as argyria. METHODS: Contents of three company websites selling colloidal silver were reviewed, and the claims used in the marketing of colloidal silver were compared to the scientific information about silver. In Facebook posts and discussion about colloidal silver were analyzed. RESULTS: In Finland, the marketing of colloidal silver products on websites selling the products did not follow the regulations of authorities; several scientifically unfounded claims about the efficacy and medical use of colloidal silver were found. After the Finnish Broadcasting Company (Yle) documentary and an intervention by authorities, contents of the websites were changed, but still questionable information and misleading claims could be found. In the analyzed Facebook groups attitudes towards medical use of colloidal silver were uncritically positive, internal use was highly promoted and the restrictions of use were considered unjustified. CONCLUSIONS: The use of quackery products such as colloidal silver can be dangerous, and their use and marketing should be controlled and restricted.

Chained Risk Assessment for Life-Long Disease Burden of Early Exposures - Demonstration of Concept Using Prenatal Maternal Smoking.

Traditional risk factors and environmental exposures only explain less than half of the disease burden. The developmental origin of the health and disease (DOHaD) concept proposes that prenatal and early postnatal exposures increase disease susceptibility throughout life. The aim of this work is to demonstrate the application of the DOHaD concept in a chained risk assessment and to provide an estimate of later in life burden of disease related to maternal smoking. We conducted three systematic literature searches for meta-analysis and reviewed the literature reporting meta-analyses of long-term health outcomes associated with maternal smoking and intermediate risk factors (preterm birth, low birth weight, childhood overweight). In the chained model the three selected risk factors explained an additional 2% (34,000 DALY) of the total non-communicable disease burden (1.4 million DALY) in 2017. Being overweight in childhood was the most important risk factor (28,000 DALY). Maternal smoking was directly associated with 170 DALY and indirectly via the three intermediate risk factors 1000 DALY (1200 DALY in total). The results confirm the potential to explain a previously unattributed part of the non-communicable diseases by the DOHAD concept. It is likely that relevant outcomes are missing, resulting in an underestimation of disease burden.

Effects of maternal smoking on body size and proportions at birth: a register-based cohort study of 1.4 million births.

OBJECTIVES: The aim of our work was to analyse the effect of maternal smoking on body size and body proportions of newborns when the mother had smoked only during the first trimester, in comparison with continued smoking after the first trimester. Furthermore, we have evaluated how growth restriction associated with maternal smoking contributes to changes in body proportions. DESIGN: Register-based cohort study SETTING: Maternal Exposure (MATEX) cohort identified from the Finnish Medical Birth Register. PARTICIPANTS: Singleton births without congenital anomalies and missing data (1.38 million) from 1 January 1991 to 31 December 2016. METHODS: Logistic regression was used to quantify the effect of maternal smoking, stratified by the maternal smoking status. OUTCOME MEASURES: Body proportions indicated by low brain-to-body ratio (defined as <10th percentile); high ponderal index and high head-to-length ratio (defined as >90th percentile); small body size for gestational age at birth (defined as weight, length or head circumference <10th percentile) and preterm birth (<37 weeks) and low birth weight (2500 g). RESULTS: Continued smoking after the first trimester was associated with high ponderal index (OR 1.26, 95% CI 1.23 to 1.28), low brain-to-body ratio (1.11, 1.07-1.15) and high head-to-length ratio (1.22, 1.19-1.26), corresponding with absolute risks of 22%, 10% and 19%, respectively). The effects were slightly lower when smoking had been quit during the first trimester. Similar effects were seen for the body size variables and low birth weight. Preterm birth was not associated with smoking only during first trimester. CONCLUSIONS: Maternal smoking, independent of smoking duration during pregnancy, was associated with abnormal body proportions resulting from larger reduction of length and head circumference in comparison to weight. The effects of having quit smoking during the first trimester and having continued smoking after the first trimester were similar, suggesting the importance of early pregnancy as a sensitive exposure window.

Smoking during pregnancy in Finland - Trends in the MATEX cohort.

Aims: In Finland, smoking rates in the general population are decreasing due to increased awareness of the adverse effects and tightened tobacco legislation. However, previous studies have shown that smoking in pregnant Finnish women remained as high as in the general Finnish female population at around 15% in 2010. Our aim was to describe temporal and spatial trends in smoking behaviour, and determinants of changes in smoking behaviour between first and second pregnancy. Methods: Self-reported smoking from the Finnish Medical Birth Register covered the years 1991-2015 (N=1,435,009). The association of maternal age and socioeconomic status with smoking rate was analysed. Spatial trends were assessed at municipality level. Results: The overall smoking rate during early pregnancy remained fairly stable at around 15% from 1991 to 2015, but increased in teenage and young women below 25 years of age. The mean smoking rate (36%) was higher in these age groups than in older pregnant women (11%). Through the study period the smoking rate remained higher in blue collar workers compared with higher socioeconomic groups. Between the first and second child, on average only 4% of women started to smoke and 41% quitted. Smoking rates developed less favourably in Eastern Finland. Conclusions: The observed increase in smoking rate during pregnancy in teenage and young women is concerning. Pregnancy is a trigger point for smoking cessation in a big fraction of pregnant women. More studies are needed to explain the opposite trends of smoking rates in Northern and Western Finland compared with Eastern Finland.

Behavioral testing of mice exposed to intermediate frequency magnetic fields indicates mild memory impairment.

Human exposure to intermediate frequency magnetic fields (MF) is increasing due to applications like electronic article surveillance systems and induction heating cooking hobs. However, limited data is available on their possible health effects. The present study assessed behavioral and histopathological consequences of exposing mice to 7.5 kHz MF at 12 or 120 µT for 5 weeks. No effects were observed on body weight, spontaneous activity, motor coordination, level of anxiety or aggression. In the Morris swim task, mice in the 120 µT group showed less steep learning curve than the other groups, but did not differ from controls in their search bias in the probe test. The passive avoidance task indicated a clear impairment of memory over 48 h in the 120 µT group. No effects on astroglial activation or neurogenesis were observed in the hippocampus. The mRNA expression of brain-derived neurotrophic factor did not change but expression of the proinflammatory cytokine tumor necrosis factor alpha mRNA was significantly increased in the 120 µT group. These findings suggest that 7.5 kHz MF exposure may lead to mild learning and memory impairment, possibly through an inflammatory reaction in the hippocampus.

The MATEX cohort - a Finnish population register birth cohort to study health effects of prenatal exposures.

BACKGROUND: The prevalence of chronic diseases, such as immune, neurobehavioral, and metabolic disorders has increased in recent decades. According to the concept of Developmental Origin of Health and Disease (DOHaD), developmental factors associated with environmental exposures and maternal lifestyle choices may partly explain the observed increase. Register-based epidemiology is a prime tool to investigate the effects of prenatal exposures over the whole life course. Our aim is to establish a Finnish register-based birth cohort, which can be used to investigate various (prenatal) exposures and their effects during the whole life course with first analyses focusing on maternal smoking and air pollution. In this paper we (i) review previous studies to identify knowledge gaps and overlaps available for cross-validation, (ii) lay out the MATEX study plan for register linkages, and (iii) analyse the study power of the baseline MATEX cohort for selected endpoints identified from the international literature. METHODS/DESIGN: The MATEX cohort is a fully register-based cohort identified from the Finnish Medical Birth Register (MBR) (1987-2015). Information from the MBR will be linked with other Finnish health registers and the population register to link the cohort with air quality data. Epidemiological analyses will be conducted for maternal smoking and air pollution and a range of health endpoints. DISCUSSION: The MATEX cohort consists of 1.75 million mother-child pairs with a maximum follow up time of 29 years. This makes the cohort big enough to reach sufficient statistical power to investigate rare outcomes, such as birth anomalies, childhood cancers, and sudden infant death syndrome (SIDS). The linkage between different registers allows for an extension of the scope of the cohort and a follow up from the prenatal period to decades later in life.

Skeletal and dental effects on rats following in utero/lactational exposure to the non-dioxin-like polychlorinated biphenyl PCB 180.

Polychlorinated biphenyls (PCBs) are a large class of persistent organic pollutants that are potentially harmful to human and wildlife health. Although a small number of dioxin-like (DL) PCBs are well characterized, the majority of PCBs have non-dioxin-like (NDL) modes of action and biological effects that are less understood. We conducted a dose-response study of the skeletal and dental effects of in utero/lactational exposure to 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB 180), a NDL PCB congener that is abundantly present in the environment and foods, including mother's milk. In a sample of 35- and 84-day-old male and female offspring from pregnant rats exposed to different doses of PCB 180 (0, 10, 30, 100, 300, and 1000 mg/kg bw), we measured the three-dimensional (3D) coordinates of 27 landmarks on the craniofacial skeleton with a Microscribe G2X system, the buccolingual width of all molars with digital sliding calipers, and a variety of tibial parameters with peripheral quantitative computed tomography (pQCT) and a biomechanical testing apparatus. The landmark coordinates were analyzed for variation in size, shape, and fluctuating asymmetry (FA) using MorphoJ software, showing no effects on cranial size, on FA in females only (i.e., decreased asymmetry), and on shape in both sexes (i.e., decreased facial length and shift in the palatal suture). In the maxillary teeth, females in the highest dose group showed a significant decrease of 0.1 mm (p = 0.033) of the second molar only, whereas males in most dose groups showed average increases of 0.1 mm (p = 0.006-0.044) in all three molars. In the mandibular teeth, the only significant response to PCB 180 exposure was the average increase of 0.1 mm (p = 0.001-0.025) in the third molars of males only. Males also shower greater sensitivity in postcranial effects of increased tibial length and decreased cortical bone mass density, although only females showed significant effects on tibial bone area and thickness. These results demonstrate marked sex differences in effects of PCB 180, which can be attributed to differences in their underlying biological mechanisms of toxicity. Furthermore, although tooth and bone development are targets of both DL and NDL compounds, this study shows that there are marked differences in their mechanisms and effects.

TCDD-induced mitochondrial superoxide production does not lead to mitochondrial degeneration or genomic instability in human SH-SY5Y neuroblastoma cells.

Several genotoxic and non-genotoxic agents have been reported to cause delayed genetic damage in the progeny of the exposed cells. Such induced genomic instability (IGI) may be a driving force in carcinogenesis, and it is thus highly important to understand the cellular events accompanying it. The aim of this study was to investigate whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects mitochondrial integrity and can consequently induce genomic instability. Mitochondrial integrity was evaluated by measuring mitochondrial superoxide production, mitochondrial membrane potential, and mitochondrial activity. Micronucleus formation was used to assess immediate genetic damage and IGI. The assays were performed either immediately, 8 or 15d after the exposure. Mitochondrial superoxide production was increased by TCDD immediately after the exposure. No consistent effects on mitochondrial integrity were observed at later time points, although slightly decreased mitochondrial membrane potential at 8d and increased mitochondrial superoxide potential production at 15 after exposure were observed in the TCDD-exposed cells. TCDD did not cause immediate genetic damage, and significant IGI was not observed. In conclusion, the present results suggest that immediate TCDD-induced increase in mitochondrial superoxide level does not lead to persistent loss of mitochondrial integrity or IGI in human SH-SY5Y neuroblastoma cells.

Maternal Smoking and the Risk of Cancer in Early Life - A Meta-Analysis.

BACKGROUND: In spite of the well-known harmful effects on the fetus, many women continue smoking during pregnancy. Smoking as an important source of toxic chemicals may contribute to the developmental origin of diseases. OBJECTIVES: The aim of this work was to pursue the possible association between maternal smoking and cancer in early life. Specifically, we wanted to identify the associated early life cancer types, and to quantify the associations. METHODS: In a systematic literature search 825 articles were identified in PubMed and Web of Science, and 55 more through the reference lists. Of these 62 fulfilled the criteria for inclusion in meta-analyses. Using Mantel-Haenszel or DerSimonian and Laird method, depending on heterogeneity of the studies, pooled estimates and 95% confidence intervals for eight cancer types were calculated. RESULTS: Smoking during pregnancy was associated with an increased risk for for brain and central nervous system tumors (OR = 1.09; 95% CI = 1.02-1.17). Although the risk for lymphoma was also associated (OR = 1.21; 95% CI = 1.05-1.34), it did not hold up in subgroup analyses. Leukemia was not found to be associated with maternal smoking. Five other cancer types (bone, soft tissue, renal, hepatic, and germ cell cancer) were also examined, but the number of studies was too limited to exclude the possibility of maternal smoking as a risk factor for cancer in offspring. CONCLUSIONS: According to our meta-analyses, maternal smoking is associated with nervous system cancers, but not with leukemia in early life. Confirming or rejecting associations of maternal smoking with lymphoma and the five other cancer types requires further studies.

Persistent organic pollutants and non-alcoholic fatty liver disease in morbidly obese patients: a cohort study.

BACKGROUND: In animal experiments persistent organic pollutants (POPs) cause hepatosteatosis. In epidemiological studies POPs have positive associations with serum markers of nonalcoholic fatty liver disease (NAFLD) and together with obesity synergistic association with insulin resistance. Because insulin resistance and obesity are critical in NAFLD pathogenesis, we investigated the association of serum pollutant levels with liver histology and alanine aminotransferase (ALT) in morbidly obese. METHODS: Liver biopsies were from 161 participants of the Kuopio Obesity Surgery Study (KOBS) who underwent bariatric surgery 2005-2011. Liver histology was categorized as normal, steatosis and non-alcoholic steatohepatitis (NASH). Liver phenotype at baseline and ALT at baseline and 12 months post-surgery were correlated to serum POP concentrations at respective time points. As lipophilic POPs concentrate to smaller fat volume during weight loss, serum levels before and 12 months after bariatric surgery were compared. RESULTS: Baseline serum concentration of PCB-118, ß-HCH and several PFAAs had an inverse association with lobular inflammation possibly due to changes in bile acid metabolism. ALT had negative associations with many POPs at baseline that turned positive at 12 months after major clinical improvements. There was an interaction between some POPs and sex at 12 months, and in stratified data positive associations were observed mainly in females but not in males. CONCLUSIONS: We found a negative association between serum concentrations of PCB-118, ß-HCH and several PFAAs with lobular inflammation at baseline. Positive POPs-ATL associations at 12 months among women suggest that increased POP concentrations may decrease the degree of liver recovery.

Hydroxyl metabolite of PCB 180 induces DNA damage signaling and enhances the DNA damaging effect of benzo[a]pyrene.

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) and their hydroxyl metabolites (OH-PCBs) are ubiquitous environmental contaminants in human tissues and blood. The toxicological impact of these metabolites is poorly understood. In this study rats were exposed to ultrapure PCB180 (10-1000mg/kgbw) for 28days and induction of genotoxic stress in liver was investigated. DNA damage signaling proteins (pChk1Ser317 and γH2AXSer319) were increased dose dependently in female rats. This increase was paralleled by increasing levels of the metabolite 3'-OH-PCB180. pChk1 was the most sensitive marker. In in vitro studies HepG2 cells were exposed to 1µM of PCB180 and 3'-OH-PCB180 or the positive control benzo[a]pyrene (BaP, 5µM). 3'-OH-PCB180, but not PCB180, induced CYP1A1 mRNA and γH2AX. CYP1A1 mRNA induction was seen at 1h, and γH2AX at 3h. The anti-oxidant N-Acetyl-l-Cysteine (NAC) completely prevented, and 17ß-estradiol amplified the γH2AX induction by 3'-OH-PCB180. As 3'-OH-PCB180 induced CYP1A1, a major BaP-metabolizing and activating enzyme, interactions between 3'-OH-PCB180 and BaP was also studied. The metabolite amplified the DNA damage signaling response to BaP. In conclusion, metabolism of PCB180 to its hydroxyl metabolite and the subsequent induction of CYP1A1 seem important for DNA damage induced by PCB180 in vivo. Amplification of the response with estradiol may explain why DNA damage was only seen in female rats.

Inhibitory effects on osteoblast differentiation in vitro by the polychlorinated biphenyl mixture Aroclor 1254 are mainly associated with the dioxin-like constituents.

The polychlorinated biphenyl (PCB) mixture Aroclor 1254 alters bone tissue properties. However, the mechanisms responsible for the observed effects have not yet been clarified. This study compared the effect of Aroclor 1254 on the expression of osteoblast differentiation markers in MC3T3-E1 cells with the corresponding effect of the dioxin reference compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and two PCB congeners belonging to the category of non-dioxin-like PCBs. The aim of the study was to quantify the relative influence of dioxin-like and non-dioxin-like PCB-components on osteoblast differentiation. Expression of marker genes for AhR activity and osteoblast differentiation were analyzed, and relative potency (REP) values were derived from Benchmark concentration-effect curves. Expression of alkaline phosphatase and osteocalcin were decreased by both Aroclor 1254 and TCDD exposure, while the PCB-congeners PCB19 and PCB52 slightly induced the expression. The relative potency of Aroclor 1254 for inhibitory effects on osteoblast differentiation marker genes was within the expected range as estimated from the chemical composition of Aroclor 1254. These results are consistent with previously observed bone modulations following in vivo exposure to Aroclor 1254 and TCDD, and demonstrate that the inhibitory effects of Aroclor 1254 on osteoblast differentiation by the dioxin-like constituents are over-riding the contribution of non-dioxin-like PCBs.

Dose- and time-dependent changes of micronucleus frequency and gene expression in the progeny of irradiated cells: two components in radiation-induced genomic instability?

Murine embryonic C3H/10T½ fibroblasts were exposed to X-rays at doses of 0.2, 0.5, 1, 2 or 5 Gy. To follow the development of radiation-induced genomic instability (RIGI), the frequency of micronuclei was measured with flow cytometry at 2 days after exposure and in the progeny of the irradiated cells at 8 and 15 days after exposure. Gene expression was measured at the same points in time by PCR arrays profiling the expression of 84 cancer-relevant genes. The micronucleus results showed a gradual decrease in the slope of the dose-response curve between days 2 and 15. The data were consistent with a model assuming two components in RIGI. The first component is characterized by dose-dependent increase in micronuclei. It may persist more than ten cell generations depending on dose, but eventually disappears. The second component is more persistent and independent of dose above a threshold higher than 0.2 Gy. Gene expression analysis 2 days after irradiation at 5 Gy showed consistent changes in genes that typically respond to DNA damage. However, the consistency of changes decreased with time, suggesting that non-specificity and increased heterogeneity of gene expression are characteristic to the second, more persistent component of RIGI.

Synergistic effects of tributyltin and 2,3,7,8-tetrachlorodibenzo-p-dioxin on differentiating osteoblasts and osteoclasts.

The purpose of this study was to examine the effects of the persistent and accumulative environmental pollutants tributyltin (TBT) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) individually and in combination on differentiating bone cells. TBT and TCDD are chemically distinct compounds with different mechanisms of toxicity, but they typically have the same sources of exposure and both have been shown to affect bone development at low exposure levels. Bone marrow stem cells were isolated from femurs and tibias of C57BL/6J mice, differentiated in culture into osteoblasts or osteoclasts and exposed to 0.1-10nM TBT, 0.01-1nM TCDD or 10nM TBT+ 1nM TCDD. In osteoblasts, the combined exposure to TBT and TCDD significantly decreased the mRNA expression of alkaline phosphatase and osteocalcin more than TBT or TCDD alone. PCR array showed different gene expression profiles for TBT and TCDD individually, and the combination evoked several additional alterations in gene expression. Expression of aryl hydrocarbon receptor repressor (AHRR) was increased by TCDD as expected, but simultaneous exposure to TBT prevented the increase thus potentially strengthening AHR-mediated effects of TCDD. The number of osteoclasts was reduced by TCDD alone and in combination with TBT, but TBT alone had no effect. However, the total area of resorbed bone was remarkably lower after combined exposure than after TBT or TCDD alone. In conclusion, very low concentrations of TBT and TCDD have synergistic deleterious effects on bone formation and additive effects on bone resorption.

Radiation-induced genomic instability in Caenorhabditis elegans.

Radiation-induced genomic instability has been well documented, particularly in vitro. However, the understanding of its mechanisms and their consequences in vivo is still limited. In this study, Caenorhabditis elegans (C. elegans; strain CB665) nematodes were exposed to X-rays at doses of 0.1, 1, 3 or 10Gy. The endpoints were measured several generations after exposure and included mutations in the movement-related gene unc-58, alterations in gene expression analysed with oligoarrays containing the entire C. elegans genome, and micro-satellite mutations measured by capillary electrophoresis. The progeny of the irradiated nematodes showed an increased mutation frequency in the unc-58 gene, with a maximum response observed at 1Gy. Significant differences were also found in gene expression between the irradiated (1Gy) and non-irradiated nematode lines. Differences in gene expression did not show clear clustering into certain gene categories, suggesting that the instability might be a chaotic process rather than a result of changes in the function of few specific genes such as, e.g., those responsible for DNA repair. Increased heterogeneity in gene expression, which has previously been described in irradiated cultured human lymphocytes, was also observed in the present study in C. elegans, the coefficient of variation of gene expression being higher in the progeny of irradiated nematodes than in control nematodes. To the best of our knowledge, this is the first publication reporting radiation-induced genomic instability in C. elegans.

Dioxin induces genomic instability in mouse embryonic fibroblasts.

Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI), i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mouse embryonic fibroblasts (C3H10T1/2) were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN) and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days). For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay), was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

Dioxins interfere with differentiation of osteoblasts and osteoclasts.

We have previously shown that the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects bone growth, modelling and mechanical strength in vivo. In this study, we utilized differentiation of bone marrow stem cells to osteoblasts and osteoclasts as a model system to study the effects of TCDD on bones. Stem cells were isolated from bone marrow of femurs and tibias of rats and mice. Progress of osteoblastic differentiation was monitored by measuring mRNA expression levels of differentiation markers from control and TCDD-treated cells using quantitative RT-PCR. TCDD significantly and dose-dependently decreased the mRNA levels of RUNX2, alkaline phosphatase and osteocalcin. Also the activity of alkaline phosphatase was significantly inhibited in both rat and mice cells. In the case of osteoclasts, TCDD decreased the number of TRACP+ multinucleated cells, with corresponding decreases in the number of F-actin rings and the area of resorption. Studies in AHR-knockout mice indicated that TCDD has no effect on the expression of osteoblastic differentiation markers suggesting that TCDD mediates its effects by AHR. Both osteoblastic and osteoclastic effects took place at very low doses of TCDD, as in most cases 100 fM TCDD was enough to significantly affect the differentiation markers. Therefore, differentiation of osteoblasts and osteoclasts from bone marrow stem cells seems to be a very sensitive target for TCDD. Disrupting effects in osteoblastic cells, in addition to disturbed osteoclastogenesis, may thus play a role in adverse effects on bone quality in TCDD exposed animals.

Differences in acute toxicity syndromes of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin in rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent congener of polychlorinated dibenzo-p-dioxins. The potency of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) is only 10% of that of TCDD for typical aryl hydrocarbon receptor (AHR)-mediated effects. Acute lethality, macroscopic effects, and liver toxicity of TCDD and HxCDD were compared in male rats of the strain Han/Wistar (Kuopio; H/W), and of the lines A and B. The latter two rat lines originate from crossbreeding of H/W and Long-Evans (Turku/AB) rats. H/W and line A rats are highly resistant to acute toxicity of TCDD due to an altered AHR, while line B rats are moderately resistant due to H/W-type alleles of another, yet unidentified gene contributing to TCDD resistance ("gene B"). The rats received 200-10,000 microg/kg of either TCDD or HxCDD intragastrically and were monitored for 46 days. In all rats, the highest dose of HxCDD (10,000 microg/kg) reduced body weight more effectively than an identical dose of TCDD. Only HxCDD (10,000 microg/kg) caused gastrointestinal hemorrhage, pale (fatty) livers and death by day 15 in H/W and line A rats. In line B rats, HxCDD caused pronounced hepatic fatty degeneration, whereas TCDD induced hepatic accumulation of biliverdin and its derivatives. Both congeners induced sinusoidal distension in liver. In H/W and line A rats, the estimated LD(50) values were >10,000 microg/kg and 2000-10,000 microg/kg for TCDD and HxCDD, respectively; for line B rats they were 480 microg/kg and 1000-2000 microg/kg, respectively. Thus, HxCDD was more potent than TCDD in inducing acute mortality in H/W and line A rats, contrary to what is predicted by toxic equivalency factor (TEF) values. In line B, the expected rank order of potencies prevailed. These findings suggest that in addition to the canonical AHR-mediated toxic pathways, HxCDD possesses an AHR-independent mechanism of toxicity, whose main manifestations are rapid body weight loss, mortality, fatty liver and gastrointestinal hemorrhage.