Genotoxicity of formaldehyde

1
Genotoxicity of formaldehyde

• Günter Speit
• Universität Ulm
• Abteilung Humangenetik
• D-89069 Ulm (Germany)
• guenter.speit_at_uni-ulm.de

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Formaldehyde is genotoxicin cultured mammalian
cells

• DNA-Protein-Crosslinks (DPX)
• Comet Assay (reduction of DNA-migration)
• Sister Chromatid Exchange Test (SCE)
• Chromosome Aberration Test
• Micronucleus Test
• Mouse Lymphoma Assay (small colonies)
• _____________________________________

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Induction of DNA-protein crosslinks by
formaldehyde
FA reacts with an amino group to form a Schiff
base (1)which can then react (2)with another
amino group(of a DNA base).
Structure of a FA-induced crosslink between
cytosine and lysine.
Ref. Barker et al. Mutat. Res. 589, 111-135
(2005)
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Formaldehyde-induced crosslinks in relationship
to cytotoxicity in V79 cells
? FA concentrations higher than 0.1 mM
significantly increasethe amount of DPX and
reduce clonal growth of V79 cells.
Ref. Merk Speit, EMM 32,260-268 (1998)
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Measurement of induction and repair of crosslinks
induced by formaldehyde with the comet assay
Reduction of gamma-irradiation induced DNA
migration by FA
Repair of FA-induced DPX
?The comet assay detects DPX in V79 cells with
high sensitivity.? Complete repair occurs within
24 hours.
Ref. Merk Speit, EMM 32, 260-268 (1998)
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Induction of sister chromatid exchanges (SCE) and
micronuclei (MN) by formaldehyde in V79 cells
? Formaldehydeefficiently inducescytogenetic
effects
Ref. Merk and Speit,EMM 32, 260-268 (1998)
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The effect of formaldehyde on mutant
frequenciesin mammalian cell gene mutation tests
HPRT-test
TK/- - MLA
? Formaldehyde does not efficiently induce gene
mutations
Refs. Merk Speit, EMM 32, 260-268 (1998)
Speit Merk, Mutagenesis 17, 183-187 (2002)
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SummaryGenotoxicity of formaldehyde in vitro

• FA-induced DPX seem to be related to the
  formation of cytogenetic effects (SCE,
  chromosomal aberrations and micronuclei).
• FA does not efficiently induce gene mutations.
• ? Chromosomal aberrations or micronuclei
  seem to be the most relevant genetic endpoints
  for studying mutagenic effects of FA in vivo.

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Systemic genotoxicity of formaldehyde in vivo

• No induction of DPX in the bone marrow of rats.
• No induction of DPX in the bone marrow of
  monkeys.
• No induction of chromosomal aberrations in
  the bone marrow of rats (one positive study
  considered inconclusive).
• No induction of chromosomal aberrations and
  micronuclei in the bone marrow of mice.
• Conflicting results in human studies
  (lymphocytes).

Review Heck Casanova, Regulat. Toxicol.
Pharmacol. 40, 92-106 (2004)
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Examples of studies indicating systemic genotoxic
effectsof formaldehyde in peripheral blood
lymphocytes of humans
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EUROPEAN COMMISSIONTechnical Guidance
Documentson Risk Assessment (TGD)
? Human data and their relevance have to be
assessed carefully on a case-by-case basis
due to limitations of the techniques
available. In particular, attention should
be paid to the adequacy of the exposure
information, confounding factors, and to
sources of bias in the study design.
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SummarySystemic genotoxicity of formaldehyde in
vivo

• FA does not induce DPX and cytogenetic effects
  in the bone marrow of experimental animals
  after high exposure.
• Conflicting results exist with regard to
  effects on PBL of human subjects after
  FA-exposure. Positive studies lack validity
  and plausibility.
• There is no convincing evidence that FA
  induces distant-site genotoxicity detectable
  with standard genotoxicity tests.

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Local genotoxicity of formaldehyde in vivo

• ? Due to the high reacticvity of
  formaldehyde,the reported local genotoxicity at
  the site of contactis particularly important
• DNA-Protein-Crosslinks (DPX) in the upper
  respiratory tract of rats and monkeys
• Micronuclei in nasal / buccal cells of humans
• _________________________________

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Local genotoxicity of formaldehyde in vivo

• Induction of DPX after inhalation (0.3 10 ppm
  6 h) in nasal mucosa cells of rats. (Casanova
  et al., 1989)
• Induction of DPX after inhalation (0.7 6 ppm 6
  h) in the upper respiratory tract of rhesus
  monkeys. (Casanova et al., 1991)

?Low FA-concentrations can induce local
genotoxicity (DPX) in experimental animals.
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Local genotoxicity of formaldehyde in humans
?The micronucleus test (MNT) has mainly been
used for monitoring exposure to formaldehyde
in exfoliated epithelial cells

• Micronuclei
• originate from chromosome fragments or
  lagging whole chromosomes
• are a sensitive indicator of damage to
  chromosomes or the mitotic spindle
• occur in G1-phase cells after cell division
  (i. e. in proliferating cells of the mucosa)

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Features of healthy mucosa

• ? parakeratinised epithelium (surface layer)
• ? prickle cell layer
• ? basal layer
• ? rete pegs
• lamina propria (underlying connective
  tissue)

Formation of MN takes place in the basal layer
where cells undergo mitosis.Induction of MN is
found in exfoliated cells several days later.
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The micronucleus tests (MNT)with exfoliated
epithelial cells

• Advantages
• relevant and well defined genetic endpoint
• relevant target cells
• simple and fast test
• no cell culture (interphase cells)
• many cells available
• cells can be fixed and stored
• applicable to large sample sizes (populations)

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MN frequencies in exfoliated cells after
formaldehyde exposure
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The micronucleus tests (MNT) with exfoliated
epithelial cells

• Shortcomings
• no standardized protocol - preparation and
  staining - evaluation (number of cells
  statistics) - interpretation (cell kinetics
  positive control)
• lack of quality criteria - cell collection
  - cell characterization (epithelial cells vs.
  leucocytes viable vs. degenerated cells)
  - reproducibility of test results
• interindividual variability of background MN
  frequencies
• large intraindividual variability for repeated
  tests

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MN-frequencies in exfoliated cells after
formaldehyde exposure

• CONCLUSIONS
• Most studies indicate induction of micronuclei
  in exfoliated cells after formaldehyde
  exposure.
• The methodology lacks standardization and
  validation.
• The results lack consistency and plausibility.
• The biological relevance of the results is
  unclear.
• The results are not suited for the
  establishment of dose-response relationships or
  for the definition of lowest observed
  adverse-effect levels.

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Genotoxicity of formaldehydeFuture directions
Future investigations should consider ? the
contribution of endogenously formed FA to
the formation of DPX, ? the genetic consequences
of DPX induced in the low dose range, ? the
relationship between the induction DPX and
the formation of mutations (e. g. micronuclei), ?
the dose-effect relationship for the formation
of mutations (e. g. micronuclei) in target
tissues.