Human risk assessment of dioxins and PCBs; uncertainties and mechanistic complexities A G Smith MRC Toxicology Unit Leicester University
Environmental polyhalogenated dioxins,furans and dioxin-like PCBs O O O Dioxins Furans Biphenyls Human exposure episodes noted from 1940s First shown experimentally as toxic in the 1970s
Dioxins etc continue to to be be of of public concern In humans exposure is of 3 types: Accidental high exposures (eg Seveso) Exposed workers in pesticide plants etc Exposure in food (main UK source) Experimental toxicity massively studied: now there is so much information it can be difficult to see the wood for the trees
Dioxin exposure in in humans COT Chloracne- most proven association but no dose response relationship Hepatic toxicity- increase in liver enzymes in plasma Cardiovascular diseases- in occupational exposure Diabetes- no consistent findings Reproductive- high exposure, changes in sex ratio; endometriosis variable Neurological/psychological- possible but variable Neurobehavioural- possible Immunological- no consistent findings Cancer- regarded as probable human carcinogen but conclusion based on overall picture from experimental and human evidence-still contentious
Chloracne is is the foremost proven consequence of of dioxin for humans; occurs at at acute high exposure (favoured by by some Ukranians) Can occur through oral exposure Rodriguez-Pichardo et al 1991 Most species are resistant Rabbits and some mice can be be made to to respond
May persist for many years, despite fall in dioxins, and not just chloracne but also other effects such as liver damage
Dioxin toxicity in in animals COT Lethality- LD50s vary from 1-5000 µg/kg Wasting syndrome- but also oedema Hepatic toxicity- includes porphyria Immunotoxicity- reduction in humoral response and splenic and thymus atrophy Thyroid hypertrophy Reproductive- deceased fertility, decreased sperm counts, effects on testes and prostate, teratogenicity Genotoxicity- mostly negative; does not bind to DNA; nonmutagenic Cancinogen- Thyroid and liver tumours, promoter of skin and liver cancer
Toxicity in in mice Mouse strains were known to respond markedly differently to polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, benzo[a]pyrene and dibenz[ah]anthracene biochemically and in cancer. This was postulated to be mediated by a receptor the Aromatic (Aryl) Hydrocarbon Receptor. Soon observed that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was the most high affinity ligand for this protein and overcame resistance in mice that did not respond to 3-MC etc. The AH receptor is a ligand activated transcription family of the PAS family. Occurs in most species (including rats and humans) but in some does not bind ligands. Subsequent studies demonstrate that most toxicities are mediated by AHR. Polymorphisms can affect efficiency of binding and thus susceptibility.
A key discovery was that dioxin (TCDD) was the most powerful ligand for the Aryl Hydrocarbon Receptor (first identified with chemicals such as benzo[a]pyrene and 3-MC) TCDD (Dioxin) Cytosol Feedback inhibition? AHRR ARA9 ARNT HIF1α DNA Hsp90 AHR NF-ΚB RB DRE (TNGCGTG) Many toxic effects? Ah gene battery mrna Cyp1a1/2 Cyp1b1 Gsta1 Aldh3a1 Nqo1 Ugt1a6 Nucleus How the myriad of toxicities occurs is far from clear
Risk assessment of of dioxins and dioxin-like PCBs based on on :: Relative affinities for AHR and dioxin-like action Human exposures (some controversial) Animal studies
Quantitation of AHR-mediated toxicities Experimentally, most aspects of dioxin toxicity and biochemical responses have been shown to depend on AHR using resistant and knockout mice Capabilities of dioxins, dibenzofurans and dioxin-like PCBs to bind to the AHR generally show structureactivity relationships similar to elicitation of biochemical and toxic responses Using TCDD as the gold standard this allows Toxic Equivalence Factors (TEFs) to be assigned to different congeners In turn this allows TEQs to be calculated for complex mixtures
Examples of TEFs* 2,3,7,8-TCDD 1,2,37,8-PeCDD OCDD 2,3,7,8-TCDF PCB 77 (3,4,3,4 ) PCB 126 (3,4,5,3,5 ) PCB 169 (3,4,5,3,4,5 ) 1 0.1 0.0001 0.1 0.0001 0.1 0.01 Sum of these allows estimation of TEQs *These get adjusted with more data
Toxicodynamics and toxicokinetics These chlorinated chemicals vary considerably in their tissue distribution and absorption metabolism and elimination Half-lives in mice and rats may only be weeks but TCDD 7.5 years in man. OCDD is 120 years in man. In terms of risk assessment, these have to be taken into account.
COT assessment To develop a risk assessment from the known hazard information, effects in animals or humans have to be selected that are relevant to humans at low doses. Is a threshold likely? With dioxins it was considered that most of the toxicity was mediated by the AHR i.e. a threshold was probably involved, and thus a NOEL + uncertainty factor approach was suitable. Find the lowest dose that gives a NOEL or if not a LOEL has to be identified. What is the criteria for exposure/dose level?
What to to choose for human study to to provide a NOEL/LOEL? It was concluded that the available human data was not sufficiently rigorous for establishment of a tolerable daily intake. Epidemiological did not reflect the most sensitive population seen in animal studies. Too many confounding factors in exposure assessments to be sure due to dioxins. Importantly, exposure of humans did not reflect UK situation where most likely from food. http://www.foodstandards.gov.uk/committees/cot/summary.htm
Animal studies Because COC had decided that the mechanisms of cancer (whatever these may be) were threshold based, all toxicological endpoints could be examined to find the most sensitive for TCDD toxicity and this would also cover increased cancer risk. In fact, very few studies could found that were in accord with modern criteria for identifying NOEL/LOEL. Of course many others were extremely good science for mechanistic and susceptibility interpretations but not suitable here. The most sensitive endpoints appeared to be on the developing reproductive systems of male rat fetuses exposed in utero. Despite inconsistencies between studies on some endpoints, it was considered that effects on sperm production and morphology represented the most sensitive effects that could be used for deriving a Tolerable Daily Intake. Sperm reserve in men is much less than the rat and thus may be highly relevant to humans.
What studies to use for TDI? 3 studies on sperm quality were available but none perfect A variety of exposure routes and endpoints and what dioxin levels present in tissues.
Use of body burden Rodents require higher doses (100-200-fold) to reach the same equivalent body burdens as in humans on exposure to food etc (differences in toxicokinetics etc). A consensus view is that body burden is the more appropriate parameter for comparison between species. The data of Hurst et al (2000) has given the distribution of TCDD in maternal and fetal tissue on Gestation Day 16 after single dose on D15 and chronic dosing before mating. This allows toxicodynamic and toxicokinetic estimates of maternal v fetal levels depending on dose route and timing. Using the two lowest doses a ratio of 2.5 was calculated to be used in estimates of body burden from single dose and subchronic exposure in other dosing studies.
What intake in humans would give a maternal body burden of TCDD of for example 30ng/kg? TCDD would show slow accumulation on repeated daily intake, because the half-life of TCDD in humans is very long (7.5 years) 35 35 30 Body load (ng/kg) 30 25 20 15 10 Body load (ng/kg) 25 20 15 10 5 Body burden = daily intake x bioavailability x half-life 0.693 Daily intake = body burden x 0.693 bioavailability x half-life 4-5 half-lives 0 5 0 0 10 20 30 40 50 60 70 Time in years 0 10 20 30 40 50 60 70 Time in years Stolen from JC Larsen and Andy Renwick! Body load (ng/kg) 35 30 25 20 15 10 5 Daily intake = body burden x 0.693 bioavailability x half-life Daily intake = 30 ng/kg x 0.693 0.5 x 7.5 years Daily intake = 15 pg/kg/day 0 0 10 20 30 40 50 60 70 Time in years
Calculation of TDI The study of Faqi et al (1998) was chosen as the most suitable for estimation of TDI although some problems and no NOEL but the lowest LOEL i.e. a loading dose then a maintenance dosing regime. Using the previous factors the subcutaneous dosing dosage regimen of Faqi et al was converted to a steady state maternal burden on GD16 at the LOEL. This was estimated as 33 ng/kg bw.
With the study of Faqi et al as the most suitable available for TDI estimation. An uncertainty factor of 1 was used for interspecies differences in toxicokinetics because of using body burdens not dose. An uncertainty factor of 1 was used for interspecies differences and human variability in toxicodynamics on the basis that rats may be more sensitive than humans but the most sensitive humans may be as sensitive as rats. An uncertainty factor of 3.2 for human variability in toxicokinetics to allow for increased accumulation in the most susceptible individuals (for dioxins with ½ lives less than TCDD). An uncertainty factor of 3 to allow for use of LOEL rather than NOEL Thus total of 9.6 (3 x 3.2 x 1 x 1) uncertainty factor
Using the overall uncertainty factor of 9.6 and the calculated maternal steady-state body burden from the study of Faqi et al (LOEL = 33 ng/kg/bw) gives a tolerable human equivalent maternal body burden of 3.4 ng/kg/bw. Putting this into daily intake (pg/kg/day) = body burden(pg/kg bw) x ln2 bioavailability x ½ life in days = 3400 x 0.693 0.5 x 2740 (7.5years) = 1.7 pg/kg/day
This TDI was rounded to 2 pg WHO TEQ/kg bw per day based on developing male reproductive system and maternal body burden WHO (1998) 1-4 pg WHO TEQ/kg bw per day SCF 14 pg WHO TEQ/kg bw per week JECFA 70 pg WHO TEQ/kg bw per month COT considered is adequate to protect against cancer and cardiovascular effects. UK consumer levels are falling but TDI is near the the value for the average consumer and lower than 97.5 percentile
Is Is this it? End of of story There actually many uncertainties 1. Dose-additivity is fundamental to the TEF idea and a reasonable idea; however we are still far from sure that this applies in the complex mixtures pertinent to human exposures. Dose reponses. 2. TEFs are mostly derived from animal data, are they appropriate for humans? e.g. carcinogenicity
The AH receptor 3. There is a lot we do not understand about the AH receptor. Ligand binding activities may not be all. Human and rat polymorphisms may affect action as a transcription factor in ways we do not understand yet. 4. Other PAS factors such as HIF and ARNT may have crucial roles in mediating toxicity. 5. What about interaction between ligands? 6. Dependence on AHR is not the same as susceptibility
Most sensitive individuals 7. What does this mean. Experimental studies are starting to show modifier genes of Ahr. Similar effects in most human diseases thought to be monogenic. QTLs for Porphyria compared to DBA/2 Dioxin C57BL/6 Dioxin SWR 11 12 14 1 9 11 HCB C57BL/10 12 14 17 For the same phenotype (porphyria) some loci in common, others different Depends on genetic background and system This may explain variability with HCB and dioxin in human exposures
Chloracne occurs at at acute high exposure Some evidence for genetic susceptibility Hairless mice are sensitive Interaction between Hr Hrgene and Ahr
What are the critical events leading to toxicity? 8. We need to pursue fundamental mechanisms to really understand the risks. What is dependent on a threshold? What influence does CYP1A2 hepatic expression have?
Other AHR ligands 9. What about non chlorinated ligands such as PAHs and natural ligands. Should we not ignore them? 10. What about chemicals such as hexachlorobenzene and brominated diphenyl ethers? All in environment. Many toxic actions are similar to dioxin-like PCBs. 11. Consideration of these aspects may cause TDI to be exceeded but?
Thanks To Diane Benford and ex colleagues on COT