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time for experts to weigh in on EFSA dismissal of Kun Lu and James A. Swenberg 2012 study that methanol becomes formaldehyde adducts inside rat cells: Rich Murray 2013.12.30

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  • Rich Murray
    time for experts to weigh in on EFSA dismissal of Kun Lu and James A. Swenberg 2012 study that methanol becomes formaldehyde adducts inside rat cells: Rich
    Message 1 of 1 , Dec 30, 2013
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      time for experts to weigh in on EFSA dismissal of Kun Lu and James A. Swenberg 2012 study that methanol becomes formaldehyde adducts inside rat cells: Rich Murray 2013.12.30

      [ See also:

      confirms WC Monte paradigm: ingested methanol becomes toxic formaldehyde-induced hydroxymethyl DNA adducts in all tissues in rats, sensitive C13 test, Kun Lu, James A Swenberg, UNC Chapel Hill 2011.12.08 Toxicol Sci: Rich Murray 2013.01.11


      [  263 page EFSA aspartame assent -- some critical notes -- also 214 public comments and Sept. 2013 US-EPA methanol review 212 page: Rich Murray 2013.12.15


      [ My comments are often in square brackets -- I add line spacing to quoted texts to make them easier to read -- my goal is to put out an initial overall survey with some critiques to help those who want to dig in deeper... It is very encouraging that all 5 of my dense public comments were published in small print, which is easy to copy and paste as normal plain text. ]


      No where in the 263 page page aspartame report does ESFA refer to Woodrow C. Monte or his breakthrough methanol formaldehyde ADH1 enzyme toxicity paradigm, even though on December 1, 2012, he send them an entire chapter 12 on birth defects from his textbook "While Science Sleeps", now available as a $ 3 Kindle 365 page ebook at amazon.com, backed by a free online archive of 782 mostly full text medical research references.  So, the mighty work is honored by its absence...

      Doubtless, vigorous scientific debate will continue... 

      [ UK COT chronic methanol toxicity in diet, 2011 July, free full text 22 pages -- earnest critical comments, applying the WC Monte methanol formaldehyde ADH1 enzyme paradigm: Rich Murray 2013.12.09

      free full text, 245 page EFSA draft report on methanol toxicity 2013.01.08 ] 

      free full text, 212 page US-EPA methanol review 2013 Sept. ]  ]


      [ EFSA draft 2013 Jan. and final aspartame review 2013.12.10 both referred very briefly to a key study by Kun Lu et al, 2012, and in the final draft attempted a vigorous critical dismissal:

      [   "In a recent study (Lu et al., 2012) formaldehyde hydroxymethyl DNA adducts have been measured after administration of labelled [13CD4]-methanol to rats (500 and 2000 mg/kg bw/day for 5 days) in multiple tissues in a dose dependent manner. 
      This finding is in line with the known metabolism of methanol." [ page 110 ] ]
        
      [ Actually this study strongly confirms the WC Monte methanol formaldehyde ADH1 toxicity paradigm!

      confirms WC Monte paradigm: ingested methanol becomes toxic formaldehyde-induced hydroxymethyl DNA adducts in all tissues in rats, sensitive C13 test, Kun Lu, James A Swenberg, UNC Chapel Hill 2011.12.08 Toxicol Sci: Rich Murray 2013.01.11

      Opps!  I was a tad hasty... 
      The EFSA Panel has concluded that the Lu study is of little merit...
      I hope the authors soon find a suitable public venue to have a legitimate discussion about this criticism.
      I imagine they and their colleagues will continue to pursue this innovative line of research. 
      It may become common practice to call formaldehyde "formaldehyde", rather than "hydrooxymethyl".

      However, on 2013.12.10, EFSA also released a link to  the 212 page Sept. 2013 US-EPA methanol review, which twice positively accepts the key finding of the Kun Lu research:

      free full text, 212 page US-EPA methanol review 2013 Sept. 

      This is a far larger group of highly qualified experts, so their very positive acceptance  of  the Kun Lu research is worthy of respectful attention -- obviously what is needed is candid discussion among experts, with many followup studies on this vital issue. ]

      quote from the Sept. 2013 US-EPA methanol review: ]

      "However, methanol can be metabolized to formaldehyde in situ by multiple organ systems (Jelski et al., 2006; Motavkin et al., 1988; Bühler et al., 1983) and dose-dependent increases of formaldehyde DNA adducts derived from exogenous methanol exposure have been observed in multiple tissues such as liver, lung, spleen, thymus, bone marrow, kidney, and WBC (exogenous adduct levels were less than 10% of endogenous adduct levels for most organ systems; embryonic tissue was not examined) of rats (Lu et al., 2012)."  ]


      [ Continuing the EFSA 2013.12.10 rejection of the Kun Lu research... ]

      "In a recent study, DNA adducts were measured in several organs after oral administration to rats by gavage of the stable isotope methanol ([13CD4]-methanol) in doses of 500 mg/kg bw and 2000 mg/kg bw per day for 5 days (Lu et al., 2012). 

      The method can distinguish between adducts formed by endogenous substances and exogenous agents.

      It has already been used to study formaldehyde adducts in the nose and at distant sites after inhalation exposure to rats (Lu et al., 2010; Lu et al., 2011) and to cynomolgus macaques (Moeller et al., 2011).

      The data of the study (Lu et al., 2012) show that labelled formaldehyde arising from [13CD4]-methanol induced N2-hydroxymethyl-dG DNA adducts in increasing numbers with increasing dose in all tissues, including liver, but in particular in the bone-marrow.

      The number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl-dG adducts in all tissues of rats.

      The ratio of exogenous/endogenous dG adducts was 0.18 and 0.45 in bone-marrow and 0.056 and 0.2 in the liver after doses of 500 mg/kg bw and 2000 mg/kg bw of stable isotope methanol respectively after adjusting for isotopic effects in the metabolism of [13CD4]-methanol.

      The Panel identified some concerns about the lower number of exogenous hydroxymethyl DNA adducts measured in the analysed tissues of rats following administration of [13CD4]-methanol at 500 mg/kg bw for 5 days compared to the number of endogenous hydroxymethyl DNA adducts.

      A previous study has shown a slower metabolic conversion of deuterium-labelled methanol compared to the unlabelled one (Brooks and Shore, 1971; Kraus and Simon, 1975).

      The Panel noted that the isotope effect factor employed was the mean of several widely varied published values. 

      Furthermore, the measurements of N-hydroxymethyl-dG and dA adducts was not direct but required an in vitro reduction process with NaCNBH3 to the corresponding N-methyl derivative.

      Based on the authors’ comments, this procedure appeared not to be quantitative as the rate of reduction was in the range of 65-85 %. 

      Furthermore, deuterium could have been exchanged by the hydride during the reduction steps, which would have allowed for depletion of the labelled material and therefore led to an underestimation of the number of exogenous DNA adducts.

      The Panel noted that the measurements of endogenous N-hydroxymethyl-dA adducts in the untreated animal group and in methanol treated group at 500 mg/kg bw for 5 days were rather variable especially in the case of white blood cells, bone-marrow and brain cells.

      Furthermore, the Panel noted that adduct formation is a biomarker of exposure of organs and tissues to methanol and that a second step is necessary between DNA adduct formation and mutagenic events (Swenberg et al., 2008; Jarabek et al., 2009). 

      Moreover, the Panel noted that doses applied in the study were in the lethal range for humans.

      [ Since humans are ten to a hundred times more vulnerable to methanol formaldehyde toxicity than any other creature, adequate toxicological tests of animals have to use high enough doses of methanol to overwhelm their more capable biochemical defense systems. ]

      Therefore, the Panel considered that the methods implemented were not sufficiently robust to support the results reported, and that no conclusion could be drawn from the study." 
      [ page 113-114 ]   ]



      [ A related presentation of the Kun Lu methods and results has been available as a free full text since 2013.03.19 -- as a medical layman, I can not contribute to this debate, except to make sections of their report available here for the convenience of qualified experts. ]

      free full text

      Chem Res Toxicol. Author manuscript; available in PMC 2013 March 19.

      Published in final edited form as:
      Chem Res Toxicol. 2012 March 19; 25(3): 664–675.

      Published online 2012 January 9. doi:  10.1021/tx200426b

      PMCID: PMC3307879
      NIHMSID: NIHMS344734

      Use of LC-MS/MS and Stable Isotopes to Differentiate Hydroxymethyl and Methyl DNA Adducts from Formaldehyde and Nitrosodimethylamine

      Kun Lu, †
      Sessaly Craft, † 
      Jun Nakamura, † 
      Benjamin C. Moeller, ‡
      and James A. Swenberg †‡*
      †Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599
      ‡Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599

      * Corresponding Author:
      James A. Swenberg, D.V.M., Ph.D., 
      The University of North Carolina at Chapel Hill,
      Department of Environmental Sciences and Engineering,
      CB# 7431, Chapel Hill, NC, 27599, 

      Abstract

      Formaldehyde is a known human and animal carcinogen that forms DNA adducts, and causes mutations. 

      While there is widespread exposure to formaldehyde in the environment, formaldehyde is also an essential biochemical in all living cells. 

      The presence of both endogenous and exogenous sources of formaldehyde makes it difficult to develop exposure-specific DNA biomarkers.

      Furthermore, chemicals such as nitrosodimethylamine form one mole of formaldehyde for every mole of methylating agent, raising questions about potential co-carcinogenesis.

      Formaldehyde-induced hydroxymethyl DNA adducts are not stable and need to be reduced to stable methyl adducts for detection, which adds another layer of complexity to identifying the origins of these adducts. 

      In this study, highly sensitive mass spectrometry methods and isotope labeled compounds were used to differentiate between endogenous and exogenous hydroxymethyl and methyl DNA adducts. 

      We demonstrate that N2-hydroxymethyl-dG is the primary DNA adduct formed in cells following formaldehyde exposure. 

      In addition, we show that alkylating agents induce methyl adducts at N2-dG and N6-dA positions, which are identical to the reduced forms of hydroxymethyl adducts arising from formaldehyde. 

      The use of highly sensitive LC-MS/MS and isotope labeled compounds for exposure solves these challenges and provides mechanistic insights on the formation and role of these DNA adducts. 



      Introduction

      Formaldehyde is classified as a human and animal carcinogen according to the International Agency for Research on Cancer (IARC) 1.

      Formaldehyde can react with proteins and DNA to form corresponding protein adducts 2;3, DNA adducts 4-7 and DNA-protein cross-links 8-15. 

      The sources of formaldehyde exposure in the body can be classified into several categories.

      Inhaled formaldehyde can enter into the body through environmental exposures such as vehicle emissions, off gassing of building materials and tobacco smoke.

      At the same time, formaldehyde is endogenously produced from serine, glycine, methionine, and choline as well as being generated from metabolism of foods, drugs, chemicals and proteins by demethylation. 

      The endogenous concentration of formaldehyde in the blood of human subjects is about 0.1 mM/L 16.
      [ 16. Heck HD, Casanova-Schmitz M, Dodd PB, Schachter EN, Witek TJ, Tosun T. Formaldehyde (CH2O) concentrations in the blood of humans and Fischer-344 rats exposed to CH2O under controlled conditions.
      Am Ind Hyg Assoc J. 1985;46:1–3. ]

      Previous research has demonstrated that formaldehyde is genotoxic in a variety of test systems, causing mutations in multiple genes 17-20. 

      DNA adducts play an important role in mutagenesis and carcinogenesis.

      The structures of formaldehyde-induced DNA adducts in vitro have been known for decades 4-7. 

      Formaldehyde can typically result in
      N6-hydroxymethyl-dA,
      N2-hydroxymethyl-dG
      and N4-hydroxymethyl-dC in vitro 21-23. 

      Hydroxymethyl DNA adducts arising from formaldehyde are not stable and need to be reduced to their methyl forms for robust quantitation.

      However, alkylation is another type of DNA damage in cells, with methyl adducts being induced by a variety of alkylating agents. 

      Such methyl adducts could be mistakenly identified as reduced hydroxymethyl adducts, as their structures are identical.

      Therefore, in this study, we have applied sensitive LC-ESI-MS/MS-SRM methods to detect and differentiate hydroxymethyl and methyl DNA adducts.

      In particular, this study was designed to address several critical questions:

      Which adduct, N2-hydroxymethyl-dG or N6-hydroxymethyl-dA,

      is the primary DNA damage following direct exposure of cells to formaldehyde?

      Does metabolically formed formaldehyde also induce hydroxymethyl DNA adducts?

      If so, which adduct is the major DNA lesion formed? 

      Do formaldehyde-generating compounds also result in DNA alkylation at N2-dG and N6-dA?

      If so, how can one differentiate alkylation products from reduced hydroxymethyl DNA adducts originating from formaldehyde?

      To answer these questions, we largely rely on the use of stable isotope labeled reagents for exposure and our highly sensitive mass spectrometry methods.

      This allows us to determine the sources of DNA adducts, their chemical characterization and provide mechanistic insights on the formation of these DNA lesions.

      In this study, we demonstrate that formaldehyde-DNA adducts arising from endogenous and exogenous sources can be clearly differentiated using [13CD2]-formaldehyde and mass spectrometry.

      Our results also demonstrate that N2-hydroxymethyl-dG is the primary DNA adduct formed following formaldehyde exposure in cells. 

      No detectable amounts of exogenous formaldehyde-induced N6-hydroxymethyl-dA were found in any exposed cells.

      In addition, we have demonstrated that DNA alkylating agents induced methylation at N2-dG and N6-dA positions, which could be confused with hydroxymethyl-dG and hydroxymethyl-dA adducts after their reduction. 

      This further defines the development of formaldehyde-specific DNA biomarkers and clarifies important issues for differentiating between methyl and reduced hydroxymethyl adducts in future research.

      Discussion

      In this study, we have applied highly sensitive LC-ESI-MS/MS-SRM methods to detect and quantify hydroxymethyl and methyl DNA adducts in cells treated by different isotope labeled compounds.

      Both endogenous formaldehyde-induced N2-hydroxymethyl-dG and exogenous [13CD2]-N2-hydroxymethyl-dG arising from [13CD2]-formaldehyde were detected.

      However, we did not observe [13CD2]-N6-hydroxymethyl-dA from exogenous formaldehyde in any exposed cells.

      In addition, we have demonstrated that metabolically generated formaldehyde also induces hydroxymethyl-dG as the primary DNA adduct in cells.

      The results support N2-hydroxymethyl-dG as a sensitive DNA biomarker to evaluate formaldehyde exposure.

      We have also confirmed the formation of two types of minor DNA alkylation adducts at N2-dG and N6-dA positions in treated cells, which were not previously reported in the literature due to their low abundance.

      The utilization of [13CD2]-formaldehyde allowed us to unambiguously differentiate formaldehyde-DNA adducts originating from endogenous and exogenous sources. 

      This study offers a unique approach to investigate potential effects of exogenous formaldehyde exposure.

      However, it is well documented that deuterium can exchange with hydrogen, which is especially evident under alkaline conditions.

      Therefore, the unambiguous identification and accurate quantification of specific DNA adducts may be influenced if H-D exchange occurs in the analyzed samples.

      Our results show that short-term exposures in cell culture do not lead to significant H-D exchange, which is consistent with our previous results using 6 hour-exposed rats. 

      Therefore, peak assignment and accurate quantitation are reliable for these samples, based on transition scanning in the mass spectrometer.

      However, when analyzing the exposed samples for a longer period, or studying the DNA adducts induced by formaldehyde from metabolic formation, special caution is needed in interpreting the peaks due to potential H-D exchange, as we have seen in the cell samples after 24 h exposure.

      In addition, we have concluded that H-D exchange occurred inside of the cells instead of in the culture medium after analyzing [13CD2]-formaldehyde extracted from culture medium after 24 h exposure.

      Previous studies show that N6-hydroxymethyl-dA is a primary formaldehyde-induced DNA monoadduct 21-23.

      Surprisingly, our data did not support the formation of detectable amounts of [13CD2]-N6-hydroxymethyl-dA from exogenous formaldehyde in cell culture. 

      The detection limit of N6-methyl-dA was more sensitive than that of N2-methyl-dG, so a lack of sensitivity does not account for our inability to detect exogenous [13CD2]-N6-hydroxymethyl-dA.

      Moreover, possible H-D exchange could hinder the identification of this adduct, however, this possibility was also ruled out after calculating the area ratios of various peaks.

      In addition, endogenous adducts and internal standards were clearly resolved at the expected retention times, supporting the conversion of N6-hydroxymethyl-dA to N6-methyl-dA and that recovery of the adduct was not an issue.

      All current evidence supports that exogenous formaldehyde does not result in detectable amounts of [13CD2]-N6-hydroxymethyl-dA in either cells exposed to [13CD2]-formaldehyde or rats exposed to 10 ppm inhalation exposures to [13CD2]-formaldehyde for 6 hours or 5 days 30.

      In this study, the endogenous amounts of formaldehyde-DNA adducts in Hela cells were determined to be ~3-4 adducts/10E7 nucleosides.

      It should be noticed that formaldehyde-induced hydroxymethyl DNA adducts were measured as corresponding methyl adducts after reduction, which may underestimate the adduct amounts due to the loss of adducts during sample processing and reduction.

      In addition, we have clearly demonstrated the formation of endogenous and exogenous methylation DNA adducts at N2-dG and N6-dA positions.

      The endogenous amount of N2-methyl-dG and N6-methyl-dA is about 0.5~0.8 adducts/10E7dG or dA.

      Alkylating agents induce exogenous N2-methyl-dG and N6-methyl-dA in a linear manner, with several-fold higher amounts of N2-methyl-dG than N6-methyl-dA. 

      Moreover, these two adducts are minor alkylation products, being ~ one hundred and one thousand times lower than those of O6-methyl-dG and N7-methyl-dG.

      These observations are consistent with previous conclusion that a violation of the Swain-Scott principle, and not SN1 versus SN2 reaction mechanism, governs DNA alkylation spectra 31. 

      The formation and biological significance of N2-methyl-dG and N6-methyl-dA in mammalian cells have not been reported 32.

      Likewise, the sources of endogenous N2-methyl-dG and N6-methyl-dA adducts are unknown. 

      They could be generated by endogenous DNA alkylating species, or they could result from formaldehyde-induced hydroxymethyl DNA adducts that were reduced by cellular reducing compounds such as ascorbic acid.

      We have demonstrated that formaldehyde induces N2-hydroxymethyl-dG as the primary DNA monoadduct in cells exposed to formaldehyde or other formaldehyde-generating compounds.

      However, a previous study demonstrated that formaldehyde lead to increased amounts of N6-hydroxymethyl-dA in multiple tissues from rats exposed to N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) 28;29. 

      Well characterized pathways support that formaldehyde is released during intracellular metabolism of carcinogenic NDMA and NNK 28. 

      Thus, increased amounts of N6-hydroxymethyl-dA could be the consequence of formaldehyde formed via intracellular metabolism of these compounds 28. 

      However, this is in marked contrast to the current study’s findings and to our earlier report 26;30 where there was no detectable [13CD2]-N6-hydroxymethyl-dA in either formaldehyde-exposed cells or nasal epithelial DNA following inhalation exposure to 10 ppm [13CD2]-formaldehyde.

      Likewise, exogenous N2-hydroxymethyl-dG, but not N6-hydroxymethyl-dA, was found in HepG2 cells exposed to N-nitrosodimethyl-D6-amine. 

      The previous study did not use isotope labeled compound for exposure, which did not allow the differentiation between formaldehyde-induced hydroxymethyl-DNA adducts and alkylation products after NaCNBH3 treatment.

      Furthermore, N2-hydroxymethyl-dG was not measured in the previous study 28, but we would predict that it would be increased.

      Our data do not rule out the possibility of forming exogenous N6-hydroxymethyl-dA. 

      However, we suggest that exogenous N6-hydroxymethyl-dA, if formed, can only be induced by formaldehyde generated from metabolic formation.

      Therefore, the way formaldehyde enters the tissue (from inhalation or intracellular metabolism) may play a critical role in the formation of specific DNA adducts in metabolically active tissues.

      This is an important issue since the exposure route may determine which formaldehyde-specific DNA biomarker should be used to evaluate the risk of formaldehyde through different exposure routes 33.

      Our findings challenge the relevance of dA adducts to current risk assessment of inhaled formaldehyde, other than contributing to the number of endogenous formaldehyde adducts that could form mutations due to enhanced cell proliferation.

      We readily detected both endogenous dG and dA hydroxymethyl adducts, but only exogenous dG adducts were observed.

      What may cause this difference between dG and dA toward exogenous formaldehyde during exposure?

      Since the in vitro reactivity of dG and dA toward formaldehyde is similar, the formation of exogenous DNA adducts should not be different from direct reaction between formaldehyde and the DNA bases, so dA adducts would be expected to occur.

      We hypothesize that the difference could be a consequence of their different involvement in the formation of DPC or DNA-protein interaction and suggest that exogenous dG adducts are actually formed through DPC intermediates.

      Our previous study demonstrated that dG actively forms DPC through cross-linking with lysine and cysteine, while dA only cross-linked with cysteine and histidine in much lower amounts 34. 

      Lysine-dG cross-links are the primary DPC formed, however, they are very labile 34.

      The glutathione-dG conjugate induced by formaldehyde that was previously identified by our laboratory 8 is also not stable.

      Decomposition of these labile DNA-protein cross-links may lead to, or facilitate, the formation of dG monoadducts.

      However, we suggest that this does not occur with dA since it is much less involved in the formation of DPC.

      Under our exposure conditions, exogenous formaldehyde may first target neighboring proteins due to the higher reactivity of lysine residues 3, followed by the further condensation with DNA to form DPC.

      In this study, we have demonstrated that formaldehyde induced fewer DNA adducts in culture medium than in PBS buffer, further highlighting the high reactivity between formaldehyde and proteins.

      Thus, there is a reduced chance for exogenous formaldehyde to directly react with DNA to form adducts.

      In contrast, endogenous dA and dG monoaducts may arise from the direct attack of higher concentrations of intracellular formaldehyde, estimated to be present in μM concentrations 35.

      Finally, we cannot rule out differences in repair.

      In summary, the results of this study clearly demonstrate that endogenous and exogenous formaldehyde-induced hydroxymethyl DNA adducts can be unambiguously differentiated utilizing [13CD2]-formaldehyde and mass spectrometry.

      Moreover, we have clearly shown that N2-hydroxymethyl-dG is the primary DNA adduct formed following formaldehyde exposure, which has important implications on available biomarkers for current risk assessment of inhaled formaldehyde. 

      Hydroxymethyl and methyl DNA adducts cannot be differentiated after reduction, which could cause potentially misleading assignments for sources of these adducts.

      However, the application of isotope labeled compounds for exposure can successfully solve this problem and provide better mechanistic insights about the formation of these adducts.

      Taken together, this study clearly shows that integrating highly sensitive mass spectrometry methods and the use of stable isotope labeled compounds for exposure are extremely useful to accurately identify the origins of different DNA adducts when they are formed from endogenous and exogenous sources.


      Acknowledgments

      The authors thank Dr. Dahn L. Clemens for providing VL17A cells to conduct NDMA exposure experiments and Dr. Stephen S. Hecht for his constructive comments on the manuscript.

      Funding Support

      This work was supported in part by NIH grants P30-ES10126, P42-ES05948
      and a grant from the Formaldehyde Council, Inc.
      BCM received support from T32-ES007126.

      Abbreviations

      LC-MS/MS Liquid chromatography-Mass spectrometry/Mass spectrometry

      IARC International Agency for Research on Cancer

      dA 2-deoxyadenosine

      dG 2-deoxyguanosine 

      NDMA nitrosodimethylamine

      MMS methyl-methanesulfonate

      MNU methylnitrosourea

      LC-MS Liquid chromatography-Mass spectrometry

      LC-ESI-MS/MS-SRM Liquid chromatography-Electrospray ionization-Mass spectrometry/Mass spectrometry-Selected Reaction Monitoring

      NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

      HPLC High Performance Liquid Chromatography

      Q-TOF Quadrupole Time-of-Flight

      nano-UPLC nano-Ultra Performance Liquid Chromatography



      large color photo of Kun Lu
      Photo by Tom Fuldner

      Scientists know that formaldehyde is a
      human carcinogen. But what’s not well
      known among the public is that this important
      chemical is also naturally present in our
      own bodies – a fact that makes establishing
      safe exposure levels difficult.

      “It’s an essential chemical in every living
      cell,’’ says James A. Swenberg, DVM, PhD,
      head of the Molecular Carcinogenesis and
      Mutagenesis Lab housed in UNC’s Gillings
      School of Global Public Health. “This seems
      to be lost on our regulatory agencies.”

      While working with the Chemical Industry
      Institute of Toxicology in 1980, Swenberg
      helped discover that formaldehyde caused nasal
      cancer in rats. Today, his lab produces data to
      help regulators make science-based decisions
      about safe levels of formaldehyde exposure.

      Formaldehyde is an important issue in
      North Carolina because the chemical is
      commonly used to produce furniture and
      textiles – two of the state’s
      historically largest industries
      – which means that many
      workers have been exposed to
      it. Other sources include cigarette
      smoke, auto exhaust and
      cooking fumes.

      Government issued
      trailers provided to victims of Hurricane
      Katrina also were found to have high
      levels of formaldehyde. The Environmental
      Protection Agency is now working to establish
      rules to set levels of exposure.

      Kun Lu, a doctoral student in Swenberg’s
      lab, developed a formaldehyde biomarker – a
      way to measure the amount in the body. In
      the lab, Kun exposed animals to two types
      of formaldehyde molecules. Using mass
      spectrometry, he examined whether the molecules
      had an effect on distant tissues.

      So far, their research has not shown that
      formaldehyde migrates to distant tissues in
      the body – which means there is less chance
      that it causes cancer anywhere other than in
      nasal cavities.

      “We want to see if we can find it in the
      liver or the bone marrow,” Swenberg says.
      “We don’t know what the answer is. We just
      want to put some good science behind it.”

      For his work, Lu won a 2009 Impact
      Award, given to UNC graduate students
      whose research provides special benefit to
      North Carolinians. In the recommendation
      letter to Impact Award judges, Swenberg
      wrote “Kun’s research…will strongly drive
      cancer risk assessment for North Carolina,
      the USA, and the world.” 

      – By Sylvia Adcock    ]
        


      [ James A. Swenberg <jswenber@...>, 
      Kun Lu <kunlu@...>,
      Benjamin C. Moeller <benmoeller@...>,
      Avram Gold <golda@...>,
      Louise M Ball <lmball@...>, 
      Yi Zhang <yi_zhang@...>,
      HongYu Ru <ru@...>,
      Jun Nakamura <ynakamur@...>,
      Gunnar Damgård Nielsen <gdn@...>, 
      Lisa A. Bailey <lbailey@...>,
      Lorenz R. Rhomberg <lrhomberg@...>, 
      Clarke G. Tankersley <ctankers@...>,
      Steven S. Hecht <hecht002@...>,
      Aglaia Pappa  <apappa@...>,
      Hermann M. Bolt <bolt@...>, 
      Carina Ladeira <carina.ladeira@...>, 
      Luoping Zhanga <luoping@...>, ]



      the first to dig the mine get to share the gold -- 7 recent similar animal research studies indicating much lower aspartame and methanol ADI levels: Rich Murray 2013.12.23


      Aspartame-induced apoptosis [cell death] in PC12 cells, Masaaki Kurasaki et al, Hokkaido U, Japan, Environ Toxicol Pharmacol 2013 Dec: Rich Murray 2013.12.27


      Aspartame: The hidden danger [methanol/formaldehyde] in our midst and how it kills us, 12 page review of While Science Sleeps text (Woodrow C Monte), International Health News, whole June issue, Editor: William R Ware PhD: Rich Murray 2012.06.08


      Table 5.2 is the key chart -- ADH1 enzyme at high levels in 20 tissues in body and fetus makes methanol into formaldehyde right inside cells, initiating over 20 human diseases, with full text references, WC Monte paradigm: Rich Murray 2013.03.21


      "As a matter of course, every soul citizen of Earth has a priority to quickly find and positively share evidence for healthy and safe food, drink, environment, and society."


      within the fellowship of service,

      Rich Murray,
      MA Boston University Graduate School 1967 psychology,
      BS MIT 1964 history and physics,
      254-A Donax Avenue, Imperial Beach, CA 91932-1918,






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