1. G. Speit Genotoxicity of formaldehyde in vitro and in vivo · CEFIC-2012 Günter Speit 2...

CEFIC-2012 Günter Speit 1

Genotoxicity of formaldehyde in vitro and in vivo

Günter Speit

Universität Ulm

Institut für Humangenetik

D-89069 Ulm (Germany)

[email protected]


Genotoxicity of formaldehyde in vitro and in vivo

Outline of the presentation:

I. In vitro genotoxicity / mutagenicity

and mode of action

II. In vivo local genotoxicity / mutagenicity

(site of contact)

III. In vivo systemic genotoxicity / mutagenicity


I. In vitro genotoxicity / mutagenicity of formaldehyde

Positive results in standard in vitro genotoxicity tests

with mammalian cells:

� Comet Assay (DPX)

� Sister Chromatid Exchange Test (SCE)

� Chromosome Aberration Test

� Micronucleus Test

� Mouse Lymphoma Assay


Formaldehyde (FA) induces DNA damage

� DNA-protein crosslinks (DPX)

� DNA-DNA crosslinks

� DNA monoadducts

� DPX are assumed to be the most relevant

primary DNA alterations induced by FA.

� DPX are generally measured by indirect methods

(e.g., HPLC, K-SDS assay, comet assay).


Detection of FA-induced DPX by the Comet assay

� DPX inhibit DNA migration

� Generally, inhibition of DNA migration

induced by irradiation is measured.

� Among the substances studied so far, FA is the most efficient

inhibitor of DNA migration in the comet assay.

� Detection of FA-induced “DNA strand breaks“

by the comet assay is inexplicable (assay variability,

preparation artifacts, exposure to other genotoxins).

2 Gy 2 Gy + FA


Induction of DPX by FA in the Comet assay

Speit et al., Mutagenesis 22 (2007)

A comprehensive characterization

of genotoxic effects induced by FA

in V79 cells clearly demonstrated:

� Induction of DPX by FA at conc. ≥ 25 µM

� Strong effects at high conc. (≥ 200 µM)

� No effect at low concentrations (≤ 10 µM)

� Formaldehyde does not induce

DNA strand breaks in the comet assay


Formaldehyde induces clastogenic effects

� Chromosome aberrations

� Micronuclei

� Small colony mutations

in the Mouse Lymphoma Assay

� Formaldehyde is a clastogen


Does formaldehyde induce gene mutations?

� No induction of HPRT mutations

under standard test conditions1)

� No induction of large colony mutations

in the Mouse Lymphoma Assay2)

� Gene mutations in cancer-related genes in nasal tumors

of the rat are most likely secondary events and not directly

induced by FA3,4)

�� FA does not efficiently induce “true“ gene mutations

1) Merk & Speit, 1998; 2) Speit & Merk, 2002

3) Recio et al., 1992; 4) Meng et al., 2010


Does formaldehyde induce aneuploidy?

Speit et al., Mutagenesis 26, 805-811 (2011)

� Aneuploidy is caused by damage to the mitotic apparatus

(is not due to an interaction with DNA)

� Aneuploidy is caused by an interaction with redundant targets

(has a threshold mode of action)

� The micronucleus test (MNT) is the standard genotoxicity test for the

detection of aneugens (OECD guideline 487) for regulatory purposes.

� FA induced micronuclei in cultured human lymphocytes and A 549 cells.

� FISH analysis revealed that the vast majority of induced MN

were centromere negative, thus indicating a clastogenic effect.

�� A significant aneugenic activity of FA was excluded.


In vitro genotoxicity of formaldehyde

Conclusions:

� Formaldehyde induces genotoxic effects (DPX, SCE)

and mutations in cultured mammalian cells.

� Formaldehyde mainly induces chromosomal mutations

(clastogenic MoA).

� Chromosomal effects (chromosome aberrations, micronuclei)

are particularly suited for the investigation of FA-induced

mutagenic effects in vivo.


II. In vivo local genotoxicity / mutagenicity

Two sources of information:

� Animal experiments

� Human biomonitoring studies


Is formaldehyde (FA) a genotoxic carcinogen?

� Prolonged inhalation of FA induced nasal tumors in rats.

� Highly non-linear dose-related increase.

� Increase in tumor incidence at 6 ppm or greater.

McGregor et al.,

CRT 40,245-285 (2010)


Is formaldehyde a genotoxic carcinogen?

“Genotoxic carcinogens“ are mutagenic carcinogens:

� Mutations are the critical key events for the induction

of cancer by “genotoxic carcinogens“.

� Mutations are the critical biomarkers for cancer risk assessment

for “genotoxic carcinogens“.

�� Are FA-induced mutations the cause

for the formation of nasal tumors?


Protective mechanisms lead to a threshold

for the induction of mutations

Exposure of cells / tissues pre-lesion protection by:

� Proteins, membranes

� Metabolic inactivation

Induction of DNA damage post-lesion protection by:

� DNA repair

Replication

Fixation of mutations

Speit et al., Mutat. Res. 464,149 (2000)

�

�

�


Is formaldehyde (FA) a good candidate

for a mutagen with a threshold mode of action?

� Naturally occurring

� presence of endogenous FA levels;

background levels of FA-DNA adducts.

� Rapid metabolic inactivation

� counteracts the formation of DNA adducts

� Efficient repair of primary DNA damage

� counteracts the production of mutations


Genotoxicity and mutagenicity of FA in nasal epithelial cells

� DPX can be induced in all cell types

of the nasal mucosa exposed to FA.

� Mutations are only produced in basal cells:

- if FA reaches basal cells,

- if DPX are induced in basal cells,

- if DPX escape repair,

- if damaged basal cells proliferate.

� Different dose-response for the

induction of DPX and mutations.

�� Basal cells

�� Exposure


Transcellular transmission of FA?

Neuss et al. Mutagenesis 25, 359-364 (2010)

Co-cultivation experiments show:

FA that has entered nasal epithelial cells

- is not released into the culture medium

even from highly exposed cells

- does not induce DNA damage in other

cells in close proximity to the

epithelial cells.

�� Only nasal epithelial cells at the surface

(site of first contact)

are significantly exposed to FA.


Induction of DNA adducts by FA in the nasal mucosa of rats

Swenberg et al., Toxicol. Sci. 120 (S1) S130-145 (2011)

� Sensitive detection of

N2-hydroxymethyl-dG adducts

� Differentiation between endogenous

and exogenous DNA adducts

� Nonlinear exposure-response

� Endogenous DNA adducts

predominate at low-dose exposure

�� FA-induced mutagenic effects

should only occur at high dose levels


FA does not induce micronuclei in the nasal mucosa of rats

Speit et al. Mutation Res. 721, 127-135 (2011)

Dose

(ppm)

MNcells

(‰)*

p-value**

0 4.75 ± 2.77

0.5 2.60 ± 1.92 0.16

1.0 6.58 ± 4.96 0.60

2.0 3.70 ± 2.02 0.63

6.0 0.90 ± 0.74 0.015

10.0 4.17 ± 4.03 0.55

15.0 4.83 ± 3.56 1.00

*) Six rats / group; 2000 cells / rat; **) Wilcoxon test

� Micronuclei were scored in rat

nasal epithelial cells after exposure

to FA by inhalation for 4 weeks.

� Exposure (6 ppm and higher)

induced cell proliferation

and histopathological changes.

�� No induction of micronuclei.

Limitations of this study:

- Limited experience with the method(assay variability).

- Evaluation of the whole nasal epithelium.

- No established positive control.


Does FA induce mutations in nasal mucosa cells?

� Up to now, there is no experimental evidence

for the induction of mutations by FA in nasal mucosa cells.

� There is concern about a mutagenic activity of FA

in the nasal mucosa of humans exposed to FA

because of positive human biomonitoring studies

with the micronucleus assay.

� These studies have been critically reviewed

and their reliability has been commented*.

*) Speit and Schmid, Mutation Res. 613, 1-9 (2006)


FA does not induce micronuclei in buccal mucosa cells

after exposure for 10 days with peak exposures up to 1 ppm.

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

co1 co2 day 0 day 7 day 14 day 21

MN

[‰

]

Speit et al.,

Mutation Res. 627,

129-135 (2007)

� Inhalation study performed under GLP-like conditions

� Defined exposure over 10 consecutive working days

� 4 h exposure with peak levels up to 1 ppm

� Bicycle exercises during exposure (3 x 15 min)

� Several sampling times (up to 3 weeks after exposure)


FA does not induce micronuclei in nasal mucosa cells

after exposure for 5 days with peak exposures up to 0.8 ppm.

Zeller et al., Mutagenesis 26, 555-561(2011)

Limitations of the MNT with nasal cells:

- Method not well standardized

- Low background value

(majority of slides with 0 MN)

� Inhalation study performed

under GLP-like conditions

� Defined exposure(5 days; 4 h; 0. 8 ppm peak levels

� Bicycle exercises (3 x 15 min)

� Several sampling times


In vivo local genotoxicity / mutagenicity - current status

Conclusions:

� Formaldehyde induces genotoxic effects

at the site of first contact (nasal mucosa).

� It is unknown to what extent these DNA alterations

lead to the formation of mutations in basal cells.

� It is unknown to what extent FA-induced mutations

contribute to the formation of nasal tumors.


III. In vivo systemic genotoxicity / mutagenicity

Two sources of information:

� Animal experiments

� Human biomonitoring studies


Systemic mutagenicity of formaldehyde in animal experiments

� Fischer-344 rats were exposed to FA (0.5 – 15 ppm)by inhalation for 4 weeks under GLP conditions.

� Genotoxicity tests were performed in accordancewith international guidelines.

Results:

� No induction of DNA damage / DPX in peripheral blood(comet assay).

� No induction of SCE in peripheral blood.

� No induction of micronuclei in peripheral blood(i.e., no indication for a clastogenicor aneugenic effect on bone marrow).

Speit et al., Mutat. Res. 677, 76-85 (2009)


Induction of DNA adducts by FA in rats and monkeys

Swenberg et al., Toxicol. Sci. 120 (S1) S130-145 (2011)

� Sensitive detection of

N2-hydroxymethyl-dG adducts.

� Differentiation between endogenous

and exogenous DNA adducts.

� Exogenous adducts were not detectable

in bone marrow and blood.

�� No indication for systemic genotoxicity.

�� Results strongly support

negative genotoxicity tests.


Systemic mutagenicity of formaldehyde?

Conclusions:

� Appropriately performed animal studies indicate that FA

does not induce systemic genotoxic and mutagenic effects.

� No induction of genotoxic effects in bone marrow and blood.

� No induction of chromosome aberrations and/or aneuploidy

in bone marrow.


Human biomonitoring:

Systemic genotoxic effects of FA in humans?

Genetic endpoint published positive

DNA-protein crosslinks (K-SDS assay) 2 2

DNA strand breaks (Comet assay) 3 2

Sister chromatid exchanges (SCE) 14 6

Chromosome aberrations 10 5

Micronuclei (MN) 9 6

Genetic endpoints studied in peripheral blood

?

?

� Associations between exposure and effects repeatedly reported.

� No mechanism known to explain the exposure of lymphocytes.


FA did not induce SCE and MN in peripheral blood cells

of human volunteers exposed to FA

� 40 male non-smokers; GLP-like conditions

� Exposure to FA under strictly controlled conditions

� Exposure for 5 days with peak exposures up to 0.8 ppm

�� No effect on SCE- and MN-frequencies

�� No effect on cell proliferation (PI / NDI)

Zeller et al., Mutagenesis 26, 555-561(2011)


Can exposure to formaldehyde in vivo actually lead

to cytogenetic effects in cultured human lymphocytes?

� Increased frequencies of SCE or MN require that blood

samples contain lymphocytes with high levels of DPX

(i.e., relevant exposure in vivo).

� SCE and MN are formed in vitro during proliferation.

� DPX have to persist until replication / cell division

in cultured lymphocytes to cause the formation of SCE and MN.

For details see: Schmid & Speit, Mutagenesis 22,69-74 (2007)


DPX are repaired in cultured human blood

before cytogentic effects can be produced

0

20

40

60

80

100

120

140

160

1 h 4 h 8 h 24 h

rela

tiv

e t

ail m

om

en

t .

100 µM FA

200 µM FA

300 µM FA

Comet assay;

mean of 3 different blood samples

� Persistence of DPX until S-phase requires high damage levels.


Induction of SCE by FA in human blood cultures

0

4

8

12

16

20

0 µM 25 µM 50 µM 100 µM 200 µM

formaldehyde

SC

E / m

ito

sis

1,5%

1,7%

1,9%

2,1%

2,3%

2,5%

2,7%

2,9%

0 µM 25 µM 50 µM 100 µM 200 µM

formaldehyde

pro

life

rati

on

in

dex

**

**

� The frequency of SCE is only increased

in lymphocytes with high damage levels

at the start of the culture.

� Induction of SCE is accompanied by

reduced cell proliferation (cytotoxicity).


Induction of MN by FA in human blood cultures

**

0

2

4

6

8

10

12

14

16

18

0 µM 200 µM 250 µM

formaldehyde

MN

[‰

]

1,0

1,1

1,2

1,3

1,4

1,5

1,6

1,7

1,8

0 µM 200 µM 250 µM

formaldehyde

ND

I**

� The frequency of MN is not increased

in lymphocytes with high damage levels

at the start of the culture.

� Lymphocytes with high damage levels

proliferate but do not produce MN.


Positive results in human biomonitoring studies

with cytogenetic endpoints are not plausible

The requirements for a positive cytogenetic test are not met

in human biomonitoring of FA-exposed populations:

� Lymphocytes are generally not in direct contact with FA.

� DPX do not accumulate in lymphocytes.

� A sufficient number of cells with sufficient damage levels

cannot be obtained.

� A sufficient amount of damage does not persist during cultivation

to lead to the formation of SCE or MN.

� There is no scientific explanation for positive results

� Without a scientific explanation,

these results should not be used for risk assessment.


Possible reasons for positive biomonitoring studies

with peripheral blood

� Unidentified action / metabolism of FA

� Chance findings / assay variability

� Incorrect interpretation / statistics

� Co-exposure to other genotoxins

� Inappropriate study performance

and psychological influence


Genotoxicity of formaldehyde - current status

Summary

� FA is genotoxic and mutagenic in vitro

� FA mainly induces clastogenic effects

� FA induces genotoxic effects (DPX)

at the site of first contact

� FA does not induce systemic genotoxic/mutagenic

effects in animal experiments

� Cytogenetic effects in human biomonitoring studies

are most likely not related to FA exposure


THANK YOU

Acknowledgements:

Petra Schütz Heinz-Peter Gelbke

Regina Linsenmeyer Lan Ma-Hock

Stefanie Kühner Irmgard Weber

Jasmin Zeller Wolfgang Kaufmann

Simone Neuß Stefan Durrer

Oliver Schmid Gerhard Triebig

Josef Högel Jörg U. Müller

Financial support: CEFIC / FormaCare

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