Inhaled Formaldehyde: Exogenous and Endogenous DNA Adducts and Epigenetic Alterations of micrornas

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1 Inhaled Formaldehyde: Exogenous and Endogenous DNA Adducts and Epigenetic Alterations of micrornas James Swenberg, D.V.M., Ph.D

Rats: 15ppm formaldehyde induced 5% incidence of nasal carcinomas after 2

2 Introduction More than 2 million tons/year of formaldehyde is produced worldwide and used in a wide spectrum of applications. Therefore, formaldehyde exposures from environmental and occupational sources are quite common. Formaldehyde is a known animal and human carcinogen, causing nasal cancer. 1. Rats: 15ppm formaldehyde induced 5% incidence of nasal carcinomas after 2 year-exposure (1ppm formaldehyde caused 22% incidence). 2. Humans: sufficient epidemiological evidence that formaldehyde causes nasopharyngeal cancer in humans according to IARC Limited evidence exists to support formaldehyde inducing leukemia. 1. Strong but not sufficient evidence for a causal association between leukemia and occupational exposure to formaldehyde based on IARC ( in 26) 2. No convincing mechanism for the induction of leukemia has been identified FEMA trailers used after Hurricane Katrina 15ppm 12-month formaldehyde induced nasal tumor

3 7 Tumor Incidence and Cell Proliferation in Rats Exposed to Formaldehyde 14 Tumor Incidence (%) Tumor Incidence 24-month Study (Kerns, 1983) Tumor Incidence 24-month Study (M onticello, 1996) Cell Proliferation Study 6-month (M onticello, 199) Cell Proliferation Study 12-month (M onticello, 199) Cell Proliferation Study 18-month (M onticello, 199) Cell Proliferation (mean unit length labeling index) at Nasal Level II (fold increase over control) HCHO Concentration (ppm)

4 Formaldehyde is a ubiquitous environment pollutant, but it is also an essential metabolitein all living cells. Therefore, both endogenous and exogenous formaldehyde need to be considered in risk assessment. Formaldehyde is very reactive with proteins and DNA, leading to diverse protein adducts and DNA damage. Fate and metabolism of formaldehyde endogenous sources exogenous sources adduct formation glutathione ALDH1A1 ALDH2 S-hydroxymethylglutathione ADH3 S-formylglutathione one carbon pool glutathione S-formylglutathione hydrolase formate CO 2 +H 2 O Adapted for IARC monograph 88

5 Experimental Design Rats were exposed to 1 ppm [ 13 CD 2 ]-formaldehyde for 6 hrs/day for 1 or 5 days and sacrificed within 2 hr. Nasal mucosa, lung, liver, spleen, thymus, mononuclear WBC and bone marrow were collected for DNA adduct analysis. DNA was reduced with NaCNBH 3, hydrolyzed to nucleosides and adducts were separated by HPLC and fraction collection. 2-4 µg of DNA was used for nasal tissue, bone marrow and WBC, while 2 µg was analyzed for other tissues. Thus, 5-1-fold more DNA was analyzed from Tissues distal to the site of contact. Capillary MS/MS methods were developed for N 2 -methyl-dg (detection limit 2 amol) and N 6 -CH 3 -da (detection limit 5 amol) monoadducts. Nano-UPLC-MS/MS methods were developed for dg-dg cross-links (detection limit 6 amol). Endogenous and [ 13 CD 2 ]-adducts were measured.

6 Scheme 1. The formation of N2-hydroxymethyl-dG originating from both endogenous and exogenous formaldehyde.

7 RT: MA: A. 8 B. m/z m/z m/z m/z RT: m/z m/z m/z m/z AA: RT: 7.54 AA: m/z m/z m/z m/z Time (min) RT: MA: m/z m/z m/z m/z C. D Time (min) RT: 7.56 MA: RT: 7.54 AA: RT: 7.54 AA: RT: 7.55 MA: m/z m/z m/z m/z Time (min) RT: 7.53 MA: m/z m/z m/z m/z Time (min) RT: 7.56 MA: LC-ESI-MS/MS SRM chromatograms of N2-Me-dG in typical tissues: 1 day-exposed nasal epithelium (A), 5 day-exposed nasal epithelium (B), bone marrow (C) and spleen (D).

9 Improved Methodology LOD: 2 attomoles LOQ: 4 attomoles Instrumentation Waters NanoAcquity UPLC Waters C18 T3 Nano Flow Rate:.6 µl/min 24 minute reverse phase gradient Mobile Phases: A) Water with.1% Acetic Acid B) ACN with.1 % Acetic Acid Thermo Quantum Ultra Triple Quadrupole MS Scan Speed:.1 seconds per transition Collision Energy: 17 ev Peak Width Q1:.3 dalton Q3:.5 dalton Scan Width: 1 dalton ESI nano source positive mode

10 Intensity Endogenous m/z Exogenous m/z Dosimetry of N 2 -hydroxymethyl-dg Adducts in Nasal Epithelium of Rats RT: 1.3 RT: adducts/ 1 7 dg 9. adducts/ 1 7 dg Exposure (ppm) Exogenous adducts/1 7 dg Endogenous adducts/1 7 dg.7±.2.39± ±1.33 3* 2.±.1.19±.8 6.9±3.3 4** n Internal Standard m/z RT: fmol 5.8±.5 1.4± ± ± ± ± Time (min) 15 ppm Rat NE 15.2± ± ±.92 5 *4-6 rats combined ** 2 rats combined

11 Ratio of Exogenous to Endogenous Adducts Exogenous 3 Ratio of Exogenous Versus Endogenous Adducts Endogenous Formaldehyde Exposure Dose(ppm)

12 N 2 -hydroxymethyl-dg Adduct Half-life Study 1 ln (Exogenous Adducts/1 7 dg) -1 t 1/2 = 63 hours -2 Y= -.11x.46 R 2 = Days Hours n=5 per time point Mean ±SD

13 Non-Human Primate Study 13 CD 2 O Exposure for 2 days (6 hours/day) at 2 or 6 ppm (n=4) Cynomolgus Macaque Tissues (to date) Nasal turbinates Femoral Bone Marrow Brain Lung

14 Adduct Numbers in Primate Nasal Maxilloturinbates Exposure concentrati on Exogenous adducts/1 7 dg Endogenous adducts/1 7 dg 1.9 ppm.25 ± ± ppm.41 ± ±.53 n = 3 or 4

15 Primate Femoral Bone Marrow Endogenous and Exogenous Adducts Intensity Endogenous m/z RT: E7 312 µg DNA Intensity Endogenous m/z RT: E6 178 µg DNA Intensity Intensity Exogenous m/z Internal Standard m/z RT: Time (min) 4E4 3E Time (min) 1.9 ppm 13 CD 2 O 6.1 ppm 13 CD 2 O Intensity Intensity Exogenous m/z Internal Standard m/z RT: E4 2E6 No Exogenous Adducts Detected with 5-1 fold >DNA Note: We used ~2-3 ug for nasal tissue

16 Adduct Numbers in Primate Bone Marrow Exposure concentrati on Exogenous adducts/1 7 dg Endogenous adducts/1 7 dg 1.9 ppm nd ± ppm nd ± 3.63 n = 4

17 Application to Risk Assessment Because no [ 13 CD 2 ]-N 2 -MedG adducts were detectable in primate bone marrow, we can state that they must be below the LOD. Therefore, the LOD represents a worst case upper bound for the amount of DNA analyzed. We have assumed that the relationship between airborne formaldehyde concentration and exogenous dg adducts is linear through zero. We calculated steady state concentrations based on the adduct half life and a 24/7 exposure. Risk estimates were calculated for all data sets (rats and primates).

19 MicroRNA Study Acquired nasal maxilloturbinatesamples (stored in RNAlater) from cynomolgus macaques from the Moeller et. al. study Isolated small RNA molecules Generated Agilent mirnamicroarray using 2 controls 3 2ppm formaldehyde tissue samples 3 6ppm formaldehyde tissue samples Statistical analysis revealed 3unique mirnaswith significantly different expression in monkeys exposed to 2 ppmformaldehyde (Fold Change >= +/-1.5, ANOVA p <.5, FDR q <.1) Statistical analysis revealed 13unique mirnassignificantly differentially expressed in monkeys exposed to 6 ppm formaldehyde

20 Significance of Findings All 3 of the significantly differentially expressed mirnasin the 2 ppm group were also significant in the 6 ppm group, where fold change magnitudes were larger (dose-response) Many of the significant mirnashave known associations to cancer (based on literature searches): 4 of the 13 significant mirnaswere measured as significantly differentially expressed after 1 ppm formaldehyde exposure using human lung cancer cells in the Rageret al., 211 study published in EHP.

21 Fold Change in Cancer-related mirna * Significant in exposed group compared to controls Down-regulated by formaldehyde in Rager et al. 211

22 RT-PCR of selected mirnas with altered expression upon exposure to formaldehyde.

23 Predicted targets of formaldehyde-altered mir-125b are involved in apoptosis signaling

24 Apoptosis-related genes predicted to be targeted by mir-125b were confirmed using RT-PCR.

25 Conclusions to date Exposure-induced DNA monoadducts and cross-links only occur in nasal epithelial DNA in rats and primates. Only dg monoadducts and cross-links are formed following inhalation and in vitro exposures to formaldehyde. da monoadducts may arise from intracellular formation of formaldehyde secondary to intracellular metabolism or DPC. Endogenous DNA monoadducts (dg and da) are present in all cells and tissues. Endogenous adducts are present in fold greater amounts than exogenous adducts following 1 ppm exposures to [ 13 CD 2 ]-formaldehyde for 5 days, but 1-fold greater at ~1 ppm exposures for 1 day.

26 Conclusions to date Both cytotoxicity and genotoxicity are key events for the induction of nasal carcinoma. The sustained increase in cell proliferation that results from formaldehyde cytotoxicity fixes both endogenous and exogenous DNA adducts into heritable mutations. If a rat was placed in a FEMA trailer for 6 hours, only 91/1, formaldehyde adducts would come from the exposure. The rest would be endogenous. A 6 hr exposure of a rat to the USEPA proposed safe level of formaldehyde (.7 ppt) would induce 83/1,, adducts. The lack of exogenous formaldehyde adduct formation in bone marrow and other distant sites does not support the biologic plausibility of leukemia.

27 Future Studies We have just completed exposing rats to 2 ppm for up to 28 days. DNA adducts to establish the time to steady-state and half-life at noncytotoxic exposures DNA protein cross-links DNA methylation MicroRNAs in nasal tissue and distant tissues Human CD 34+ cells to establish endogenous adduct amounts. Human bone marrow to compare with monkey data. Human nasal turbinates to establish endogenous adduct amounts. A primate study to examine stem cells in CD 34+ primed monkeys exposed to [ 13 CD 2 ]-formaldehyde.

28 Repair of Aldehyde DNA Lesions 1 RKO (parental) FANCG ko FANCC ko 1 Survival (% control) Formaldehyde (µm) Ridpath, JR et al (27) Cancer Res 28

29 DNA adduct analysis endogenous Endogenous m/z exogenous Exogenous m/z N 2 -ethylidene-dgreduced to N 2 -ethyl-dg for stability and LC-MS/MS analysis

DNA Adduct Analysis 1. Isolate DNA using Nucleobondanion exchange columns 1. Reduce DNA with NaCNBH 3 at 37 C, 6 hours with phosphate buffer, ph 7.2 2. Digest DNA 1. Enyzmes: AP, PDE, DNAse 2.

30 DNA Adduct Analysis 1. Isolate DNA using Nucleobondanion exchange columns 1. Reduce DNA with NaCNBH 3 at 37 C, 6 hours with phosphate buffer, ph Digest DNA 1. Enyzmes: AP, PDE, DNAse 2. Tris/MgCl 2, ph 7.2, IS -5 fmol 3. 6 min with 1 min pre-incubation w/ DNAse at 37 C 3. Filter with MW cutoff (Pall 3Kd Nanosep) 4. HPLC Fraction collection, ~6 min/sample A.1% acetic acid B -.1% acetic acid in ACN 5. Dry in speedvac 6. Re-dissolve in 1 µl water, inject 5 µl mau Nucleosides DAD1 B, Sig=254,4 Ref=36,1 (BEN\119211_AA_CALCURVE \11911_AA_2.D) N 2 -ethyl-dg Fraction Collection Chromatogram Waters nanouplc and Thermo Quantum Ultra min

31 UPLC-MS/MS Chromatograms Relative Abundance Relative Abundance Relative Abundance Control TK6 Cells.1 mm [ 13 C 2 ]- RT: AA: E5 Acetaldehyde Endogenous m/z Exogenous m/z IS m/z RT: AA: Time (min) 6.E3 1.9E5 Relative Abundance Relative Abundance Relative Abundance RT: AA: RT: AA: RT: 15.7 AA: RT: AA: Time (min) 8.1E5 1.E5 2.9E5 Relative Abundance Relative Abundance Relative Abundance 2. mm [ 13 C 2 ]- Acetaldehyde RT: AA: RT: AA: RT: AA: Time (min) 5.5E5 3.4E7 2.9E5 Presence of a signal in exogenous chromatogram ( m/z) is from the natural isotopic abundance (.7%) of endogenous N 2 -ethyl-dg ( m/z).

33 Exogenous and Endogenous DNA Adducts of Acetaldehyde in AHH-1 Cells

34 Sum of Adducts y = x +.3 x x 3 5 Sum Adducts/1 7 dg Endogenous Mean 1 5e-5 1e [ 13 C 2 -Acetaldehyde] mm Polynomial Model of the Sum of Adducts. The mean of the endogenous adducts was 2.98 (green line) and the 95% prediction interval (dotted lines) of the log 1 (Total DNA adducts) are shown. Thus, we have 95% confidence that if the acetaldehyde concentration is above.2 mm, the sum of the adducts will be higher than the endogenous background. 34

35 95% Prediction Interval for Micronucleus 2 %MN = X -1.2 X X 3 Spearman rank correlation.698 (p-value 3.7e-5) 1 5 % MN Vehicle Control e-5 5e [ 13 C 2 -Acetaldehyde] mm % Micronuclei Formation versus [Acetaldehyde]. The mean % MN of the vehicle control was.61% (red dash) and the 95% prediction interval (dotted lines) are shown. Thus, we have 95% confidence that if the acetaldehyde concentration is above.35 mm, the future observation of % MN will be larger than the background of.61%. n = 3 per except for 2. mm with n = 1 35

36 The Exposome Chris Wild proposed that we should be considering the Exposome for cancer etiology. Wild, C: CEBP 14: , 25 Under this view, the assessment of exposures should not be restricted to chemicals entering the body from air, water, food, smoking, etc., but should also include internally generated toxicants produced by the gut flora, inflammation, oxidative stress, lipid peroxidation, infections, and other natural biological processes. In other words, we must focus upon the internal chemical environment arising from all exposures to bioactive chemicals inside the body More recently, Martyn Smith et. al. made similar statements. Smith, M: Chemico Biological Interactions 192: , 211 The question arises as to how to find the causes of the majority of de novo AMLs that remain unexplained. We propose that we should attempt to characterize the 'exposome' of human leukemia by using unbiased laboratory-based methods to find the unknown 'environmental' factors that contribute to leukemia etiology.

37 Steady-state Amounts of Endogenous DNA Damage Endogenous DNA Lesions Number per Cell Abasic sites 5, OHEtG 3, 7-(2-Oxoethyl)guanine 3, 8-oxodG 2,4 Formaldehyde 1,-4, Acetaldehyde 1, 7-Methylguanine 1,2 AcrdG 12 M 1 dg 6 N 2,3-Ethenoguanine 36 1N 2 -Etheno dg 3 1N 6 -Etheno da 12 Total 6, +

38 Mutations Are Biomarkers of Effect, but They Do Not Go Through Zero In contrast to most DNA adducts, mutations do not go through zero. Rather, they reach a background level that reflects the summation of mutations arising from endogenous DNA damage and repair that occurs in cells. The dose-response may be linear or nonlinear. There may be an inflection point for a dose response curve where the number of mutations increases nonlinearly above the spontaneous level, or there may be a linear increase with data points that are not significantly different from controls at lower doses. The point at which the mutations increase is where the exogenous DNA damage starts drivingthe biology that results in additional mutations.

39 7 Historical Control Data for HPRT and TK Mutations in vitro 6 5 Mutant Fraction (x1-6 ) tk locus n=87 hprt locus n=34 q1 min median max q3 1 TK6 AHH-1 Cell Line Penman and Crespi, Environ Mol Mut 1:35-6, th

40 Collaborators and Sponsors Kun Lu Ben Moeller Natalie Herr Tom Starr Leonard Collins Patricia Upton Betsy Bermudez Ed Bermudez Jacob McDonald Melanie Doyle-Eisele Andrew Gigliotti Julia Rager Rebecca Fry Formaldehyde Council FormaCare-CEFIC American Chemistry Council HamnerInstitutes for Health Sciences Lovelace Respiratory Research Institute NIEHS Superfund Basic Research Program (P42-ES 5948) NIEHS Center for Environmental Health and Susceptibility (P3 ES 1126)

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