1. What environmental factors are linked to childhood leukaemia and are they detectable epidemiologically? Denis L Henshaw H H Wills Physics Laboratory University of Bristol 2 nd CHILDREN with LEUKAEMIA Conference 29 th & 30 th April 2008 Childhood Leukaemia Causes and Prevention

2. I will consider…. Natural Background Radiation low-LET stable over time; high- LET (radon) small increase Light-at-night large increase last century Power frequency magnetic fields low average exposures but a general increase Air pollution significant increase in ultrafine/nano particles (Shah & Coleman BJC 2007; 1 - 4) Childhood leukaemia is believed to have shown a real increase last century

3. Natural background ionising radiation Childhood leukaemia risk has been extensively studied at medium and high level exposures in the Japanese Atomic Bomb survivors and fallout from nuclear weapons testing (Wakeford Oncogene 23: 6404-, 2004) Increased risk in relation to in utero exposure from obstetric X-rays has been demonstrated with acute doses of 10 mSv or lower (Doll & Wakeford BJR 70: 130-, 1997) Natural background radiation exposure rate in the UK is ~2.7 mSv y -1. HPA/NRPB

4. Risk estimates derived from these exposures suggest that natural background radiation contributes to childhood leukaemia incidence UK COMARE 4th Report (1996) page 62:

5. Natural background radiation………….. For high-LET ( -particles) the 14% link divides: ~9% 210 Po RR = 1.09 at normal 210 Po levels compared with zero 210 Po ~5% Radon RR = 1.05 at 20 Bq m -3 compared with zero radon Both high and low-LET estimates are essentially undetectable in a case-control study because: (i) of ubiquitous exposure to low-LET radiation with little place-to-place variation and (ii) for radon, too few statistics in the high vs low exposure category to provide statistical power

6. Radon–leukaemia correlations There have been a number of reports of geographical associations between radon and leukaemia: Lucie 1989, Henshaw et al 1990, Alexander et al 1990, Butland et al 1990, Lucie 1990, Muirhead et al 1991 & 1992, Cohen 1993, Richardson et al 1995, Lyman et al 1986, Collman et al 1991, Viel 1993, Forastiere et al 1992, Hoffman et al 1993, Thorne et al 1996, Kohli et al 2000, Anne-Sophie Evrard et al 2004 But few case-control studies have reported an association

7. Light-at-night (LAN) Europe in the Present Europe in the past

8. The Melatonin Hypothesis and breast cancer – Richard Stevens University of Connecticut 1987 Exposure to light-at-night or magnetic fields suppresses the nocturnal production of the natural anti-cancer agent melatonin in the pineal gland leading to increased risk of breast cancer Has much support for visible light: evidence that female night shift workers have elevated breast cancer risk (Meta-analysis of 13 studies: Megdal et al. Eur J Cancer 41: 2023-2032, 2005) Womens blood containing normal physiological concentrations of nocturnal melatonin prevents growth of MCF-7 breast tumours transplanted into rats (Blask et al. Cancer Res, 65: 1-11, 2005) 2007: IARC has classed night shift work as a Class 2A, probable carcinogen

9. Melatonin (N-acetyl-5-methoxytrptamine) Melatonin is a hormone produced in the pineal gland mainly at night Ganglion cells in eye (not visual system) signal when there is no light – pineal melatonin produced at night Powerful anti-oxidant with multiple actions, many receptor mediated By a scavenging cascade – one melatonin molecule can scavenge up to 10 ROS/RNS (Tan et al J Pineal Res. 42; 28-42, 2006)

10. Light-at-night and childhood leukaemia Melatonin protects against oxidative damage to the fetus in animals (Wakatsuki et al 1999a, b &c; Okatani et al 2001) Melatonin is highly protective of oxidative damage to the human haemopoietic systems (Vijayalaxmi et al Mutat Res: 371; 221-,1996): Four human volunteers took 300 mg melatonin, after 2 hours blood samples exposed to 1.5 Gy -radiation. 50-70% reduction in DNA damage in lymphocytes Melatonin inhibits proliferation of HL60 myeloid leukaemia cells (Rubio et al J Pineal Res 42: 131-, 2007, 42) Has not been studied but:

11. Fig. 1. Changes of maternal serum melatonin levels at night-time (solid line) or daytime (dotted line) in the normal singleton pregnancy. Serum melatonin level and its relationship to feto-placental unit during pregnancy (Nakamura et al J Pineal Res 30: 29-33; 2001)

12. Power frequency electric & magnetic fields - especially magnetic fields, MFs (Richard Boxs FIELD February 2004 Photo: Stuart Bunce, www.richardbox.com) Under powerlines MFs can be several T or evens tens of T Appliances: can be tens of T close to Average MF home levels 0.05 T Doubling of CL risk associated with 0.3/0.4 T

13. Childhood leukaemia and magnetic fields (MFs) Reported MF associations with childhood leukaemia at various stages: a)Paternal peri-conceptual - Pearce et al (Pediatr Blood Cancer, 49: 280-, 2007) b)Birth address - Draper et al (BMJ, 330, 1290-, 2005); c)Diagnosis address - Ahlbom et al. (2000) and Greenland et al. (2000) – doubling of risk above 0.3/0.4 T average exposure. More recently Kabuto et al (Int J Cancer¸ 119: 643-, 2006); a)Reduced survival in children treated for ALL - Foliart et al (B J Cancer, 94, 61-, 2006); A linear no-threshold model would suggest ~11% of CL cases linked to MFs in the UK International Agency for Research on Cancer (IARC) 2001 declares magnetic fields a Class 2B possible carcinogen Statements about precaution - WHO (2007); SCENIHR (2006); UK SAGE Report (2007)

14. Disease IARC 2002 2B* NIEHS 1999** 2B Calif 2002 2B 1. Childhood LeukaemiaYes 2. Adult LeukaemiaYes 3. Adult Brain CancerYes 4. MiscarriageYes 5. ALSYes But, stepping back from CL: Review bodies assessments of MF causation of various diseases. *Class 2B, possible carcinogen **US National Institute of Environmental Sciences See also OCarroll & Henshaw (Risk Analysis, 28(1), 225-, 2008) Evidence for MF link with Adult leukaemia, and at least as good as for childhood leukaemia. Alzheimer's disease also comparable (Garcia et al Meta Analysis 25 studies: Int J Epidemiol 2008)

15. Illnesses associated with MFs – Is there a common factor? Childhood leukaemia, Adult leukaemia, Adult brain cancer, ALS, Alzheimer's disease, Depression, Suicide, Miscarriage, Breast cancer Disruption of Melatonin Populations exposed to neighbourhood EMFs shown melatonin disruption with fields as low as 0.2 T (Review by Henshaw & Reiter, Bioelectromagnetics S7: 86-97, 2005)

16. The Radical Pair Mechanism: When two radicals created from one precursor, spin states are coupled (+½, -½) – singlet state ( ~ns) If the spin of one of the species changes, radical pair will have parallel spins – triplet state ( ~ s) +½ -½ Molecule spin = 0 Singlet radical Pair (ns) Triplet radical Pair (µs) +½ -½ Spin change +½ Separation: Free radicals Magnetic fields can increase the lifetime of free radicals - More available to damage DNA

17. Low magnetic field effects – previously degenerate energy states resolve (Timmel et al. Mol Phys 95: 71-89, 1998) Increases probability of transitions from singlet (ns) to triplet state ( s) of radicals Results in more triplets – longer lived ( s), more available to cause biological damage Experimental support in chemical systems (e.g. Liu et al Chem Commun 174-, 2005) Magnetic fields can increase the lifetime of free radicals by driving singlet spin states to triplet states

18. Biological evidence for the RP mechanism – increased lifetime of free radicals Juutilainen et al. (IJRB 82; 1-12, 2006) review of 65 in vitro & animal studies – MFs enhance the effects of known harmful agents, p<0.0001 Lupke et al. (Free Radical Res 38; 985-993, 2003) – monocyte cells from cord blood exposed to 1 mT fields release reactive oxygen intermediates

19. Childhood leukaemia & MFs apparent MF effects at ~0.4 T 50/60 Hz against the Earths static magnetic field of 50 T Similarly: Fluctuations in the Earths MF of ~0.3 T appear to be linked to acute health effects e.g. depression (Review: Palmer et al: Surv. Geophys, 27, 557-,2006 )

20. Animal navigation Homing pigeons sensitive to 10-20 nT changes in the Earths 50 T field (Wiltschko, 2005) Some amphibians have receptors in the pineal gland… But of most interest in birds is the compass in the eye (e.g. robins). Ritz et al studied the magnetic compass in robins (Nature 429: 177-, 2004) - Birds detect tiny changes in magnetic fields The compass is based on the radical pair mechanism

21. In the cryptochrome proteins visible light of particular wavelength creates free radicals whose spin states are affected by MFs. Thus, cryptochromes, detect and amplify small MF changes for transmission to the brain. Ritz et al 2000 & 2004 postulated and then identified RPM in eye Ongoing work has identified ms lifetimes of triplet state cryptochromes in garden warblers (Leidvogel et al 2007 - Oxford) University of Illinois (www.ks.uiuc.edu/Research/cryptochrome) Ritz et al (Biophys J 78: 707-, 2000); Ritz et al (Nature 429: 177-, 2004) Review by Wiltschko & Wiltschko, BioEssays, 28; 157-168, 2006 Evidence suggests the avian compass uses cryptochromes in the eye

22. Cryptochromes contd… Cryptochromes CRY1 & CRY2 proteins are expressed by the cryptochrome 1 & 2 genes – part of the family of 8 core circadian genes identified to date 50 – 70 kDa in molecular size; ~4 nm in physical size Cryptochromes are expressed in plants which can respond to magnetic fields (Review: Galland & Pazur J Plant Res 118: 371-, 2005) Cryptochromes do not necessarily need visible light to create radical pairs The circadian genes are present in all cells, but it is not known whether they express cryptochromes in cells in general to facilitate the MF effects reported by Lupke et al and others

23. Air pollution

24. Peggy Reynolds, California Health Dept: Odds ratios and 95% confidence intervals for childhood leukaemia in proximity to highest vs. lowest levels of traffic-associated exposures for twelve published studies Savitz (1989) 0.1 110100 0.5 5 50 Alexander (1996) Feychting (1998) Harrison (1999) Pearson (2000) Raaschou-Nielsen (2001) Reynolds (2001) Langholz (2002) Reynolds (2002) Crosignani (2004) Reynolds (2004) Steffen (2004)

25. Urban dM dlogDa ( gm -3 ) Aerodynamic Diameter (nm) With kind permission of Drs Rami Alfarra and Hugh Coe. Hydrocarbons (PAHs) mainly on the nano-aerosols – travel considerable distances from production site

26. Nano-particle component of air pollution Total concn (11-083nm) roadside = 53 900 cm -3 Total concn (11-1083nm) 'heavy emitter' = 241 000 cm -3

27. Inhaled nano-particles pass into the bloodstream Nano-particles (NPs) when inhaled penetrate deeply into the lung and pass into the bloodstream In the context of cardio-vascular disease, NPs release reactive oxygen species: (i) Talk by Delfino (University of California, Irvine): http://www.aqmd.gov/tao/Ultrafine_Presentations/Session4_4_Delfino.pdf (ii) Simko et al (Toxicology Lett 161: 73-, 2006) – Mono Mac 6 cells release ROS by 14 nm particles

28. Heritable mutations from air pollution Steel Mills, Hamilton Harbour Lake Ontario Somers et al PNAS 99: 15904-, 2002 & Science 304: 108-, 2004 1 km downwind of steel mills: Exposed mice – ambient air Controls – air filtered to 0.01 m (10 nm) Further controls – rural location 30 km away 1.5 – 2-fold increased heritable germ line mutations in exposed group 26 PAHs measured 1 km

29. Papers by George Knox 2005 & 6 Knox 2005a (J Epidemiol Comm Health, 59, 101-105) - Childhood cancer associated with hotspots for carbon monoxide, PM10 particles, VOCs, nitrogen oxides, benzene, dioxins, 1,3-butadiene and benzo[ ]pyrene Knox 2005b (J Epidemiol Comm Health, 59, 755-760) - Childhood cancer associated with short distances of bus stations, hospitals, railways, oil installations and industrial transport centres: 1,3-butadiene especially implicated Knox 2006 (J Epidemiol Comm Health, 60, 136-141) - Childhood cancer associated with short distances of bus stations, railway stations, railways, A,B class roads: again 1,3-butadiene especially implicated (birth and death addresses of fatal child cancers in GB between 1966-80) McKinney et al UKCCS: Occup Environ Med 60: 901-, 2003

30. Knox: Compared outward/inward migration wrt birth and diagnosis address Among migrant children, distances from each address to the nearest pollution emission hot-spot were composed. Excess of outward over inward migrations show an increased prenatal or early infancy risk. B Hazard site, e.g. Bus station 1km B D Case 1 Case 2 D Address diagnosed Birth address D B

31. Findings – 2 (2005a): Benzene is a major component of motor fuel 1,3-butadiene is largely derived from petrol and diesel exhausts Distance (km)Ratio out/in 0.38.39 0.57.65 0.74.66 Close ratios for 1,3-butadiene: Knox - conclusions Excess relative risks within 1.0 km of bus stations, hospitals, heavy transport centres and oil stations. Most significant result: RR = 12.6 for joint <0.5 km exposure to bus stations and 1,3-butadiene The associations with 1,3-butadiene, dioxins and benzo[a]pyrene are sufficiently specific to conclude that they are probably the truly active agents Size of the effects suggests 30 - 80% of childhood leukaemia and cancer could be linked to vehicle related air pollution in the UK at the in utero/early infancy stage

32. Draper et al. British Medical Journal 2005; 330: 1290-3 29,081 children with cancer, 9,700 with leukaemia 1962-1995 (322 leukaemia cases <600 m) NB: Unlike other studies, this used the birth address and not the diagnosis address

33. Lowenthal et al. Internal Medicine Journal 2007 854 patients diagnosed with LPD or MPD aged 0-94 years diagnosed in Tasmania 1972-1980 ORs for ever having lived within 50 and 300 m of a high voltage power-line compared with never residing within 300 m and for risk associated by age of exposure within 300 m. 2.06 (0.87-4.91) 1.3 (0.88-1.91) <50 m50-300 m 4.74 (0.98-22.9)1 st 5 years of life 1 st 15 years of life3.23 (1.26-8.29)

34. Charge separation and ion emission High E-field ionises the air + + Initial bare ion Forms cluster of molecules (~100 ns) + Attaches to pollutant aerosol (10-100 s) When inhaled, electrically charged aerosols could have a higher probability of depositing in the lung Corona ion hypothesis Direct fields cannot explain findings of Draper and Lowenthal distant from the power lines Indirect mechanism could be due to corona ions (Fews et al. 1999) High voltage line ionises the air and forms atmospheric small-ions Small-ions attach to pollutant aerosols, changing their charge state Increased charge on aerosols can lead to increased lung deposition

35. Selected environmental exposures: possible attributable fractions childhood leukaemia risk AgentAttributable fraction Comment Infections etc This conference Natural Background ionising radiation 34%: 20% low LET 14% high LET Overall: essentially undetectable epidemiologically Light-at-night ? Not researched Magnetic fields ~11% Overall (Using linear no-threshold model) Air pollution 30 - 80% By birth address (1 st hit: in utero?)

36. Acknowledgements Team members : Julie Close Matthew Wright James Matthews Alison Buckley Andrea Lazenby Supported by Websites: www.electric-fields.bris.ac.uk/presentations.html www.electric-fields.bris.ac.uk

37. Dose response for light Zeitzer et al. J Physiol (2000) 526, 695-702

38. Childhood leukaemia near nuclear installations UK: Sellafield – Black Report (NRPB-R171; 1984) Germany: Childhood leukaemia and all malignancies near nuclear power plants (Kaatsch et al Int J Cancer 1220: 721-, 2008; Spix et al Eur J Cancer 44: 275-284; 2008) -activity in upper right 4 tooth from a 13-year-old boy: outer enamel 210 Pb-supported 210 Po; circumpulpal 226 Ra. The problem is one of dose levels: Natural -activity in childrens teeth: 5 – 20 Bq kg -1 Plutonium in childrens teeth: ~mBq kg -1 (ODonnell et al Sci Tot Environ 201; 235-, 1997)

39. The incidence of childhood leukaemia is believed to have shown a real increase last century (Shah & Coleman BJC 2007; 1 - 4)

40. Can we square the circle with Draper et al. ? (and possibly with Lowenthal et al) Knox (2005a, b, 2006) has published three papers reporting a series of statistically significant associations and dose response information between the incidence of childhood cancer and sources of petro-chemical air pollution in Great Britain We can certainly point to powerlines, 400, 275 and 132 kV which emit sufficient corona to explain the finding of Draper et al. (2005) in terms of the corona ion model What we cannot do is claim that all powerlines in England & Wales, from 1962 – 1995 are/were in sufficient corona to account for Draper et als findings

41. Activity concentrations in teeth The outer enamel surface best harbours integrated exposure to 210 Pb/ 210 Po (James et al. 2004a Int. J. Radiat. Biol., 80(3), 185-198.). Typical levels 8 – 12 Bq kg -1 but can reach over 40 Bq kg -1 Activity concentrations of 210 Pb in foetal teeth can be 40% of those in deciduous teeth from a 10 yr-old (however, little ingrowth of 210 Po in the foetus) Activity concentrations in teeth correlate well with those in bone So, we use teeth as a maker for levels in bone and dose to bone marrow

42. Ascertaining how magnetic fields cause adverse health effects Magnetic Fields 1. Physics 4. Epidemiology 3. Human Biology 2. Chemistry Proof that exposure to power frequency MFs causes increased risk of certain illnesses, requires answers to wide ranging questions: 1.Physicists would like to know the primary interaction mechanism of MFs with biological/living systems 2.Chemists would like to know how this primary interaction affects basic chemistry 3.Biologists would like to know how a physical/chemical response to MF affects biology e.g. through DNA or cellular damage and how this may result in increased risk to health 4.Epidemiologists would like to demonstrate increased risk of a given illness with MF exposure by carrying out a series of independent epidemiological studies with sufficient resolving power to detect an effect and to obtain a robust set of positive findings such that an association between exposure and increased risk can be established. (Physical Chemistry)(Biochemistry)

43. Findings – 5 (2006) Excess relative risks within short distances of bus stations, railway stations, railways and A,B class roads A roads<0.1<0.2<0.3<0.5<1.0>1.00 - 0.30 – 0.5 Births7138626406991142160715331536 Deaths33662751983112962054800936 B/D ratio2.121.371.230.840.880.781.921.64

44. Volatile organic compounds Include: Benzene – suspected leukaemogen Benzo[ ]pyrene – known carcinogen 1,3-butadiene Dioxins

45. Findings – 1 (2005a) Benzene1,3-Butadiene No of hotspots 5981111949 No of children 13092775 Outward migration 8892194 Inward migration 419581 Ratio out/in 2.123.78

46. Properties of melatonin Unlike vitamins C & E, melatonin enters cells and can act directly on DNA By a scavenging cascade – one melatonin molecule can scavenge up to 10 ROS/RNS (Tan et al J Pineal Res. 42; 28-42, 2006)

47. Knox - conclusions The associations with 1,3-butadiene, dioxins and benzo[ ]pyrene are sufficiently specific to conclude that they are probably the truly active agents Assessment of the size of the effects suggests that up to 80% of childhood leukaemia and cancer could be linked to vehicle related air pollution in the UK at the in utero/early infancy stage

48. Papers by George Knox 2005 & 6 Knox 2005a (J Epidemiol Comm Health, 59, 101-105) - Childhood cancer associated with hotspots for carbon monoxide, PM10 particles, VOCs, nitrogen oxides, benzene, dioxins, 1,3-butadiene and benzo[ ]pyrene Knox 2005b (J Epidemiol Comm Health, 59, 755-760) - Childhood cancer associated with short distances of bus stations, hospitals, railways, oil installations and industrial transport centres: 1,3-butadiene especially implicated Knox 2006 (J Epidemiol Comm Health, 60, 136-141) - Childhood cancer associated with short distances of bus stations, railway stations, railways, A,B class roads: again 1,3-butadiene especially implicated (birth and death addresses of fatal child cancers in GB between 1966-80)