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Use of nasal and buccal cells in human biomonitoring studies for the detection of cytotoxic and DNA-damaging chemicals

Siegfried Knasmller,
A. Nersesyan, M. Misik

Cancer Research Institute, Innere Medizin I Medicine University of Vienna


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Topics
background and historical notes methodological aspects MN assays with exfoliated buccal cells MN assays with nasal cells Advantages /disadvantages in comparison to MN assays with lymphocytes Use of MN assays with exfoliated cells in human biomonitoring; the current data base Studies on FA exposure Conclusions
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Background
A variety of methods has been developed to detect exposure of humans to genotoxic carcinogens in human cells Adducts Chromosomal aberrrations

Micronuclei

Comet - Assay

OH

8 - Hydroxydeoxyguanosine
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Not all endpoints are related to cancer risks !!!! MN in lymphocytes and buccal cells reflect human cancer risks. Chromosomal aberrations in lymphocytes correlate with human cancer risks. Sister chromatid exchanges in lymphocytes: no correlation. Comets: not known !!!!! DNA Adducts: for certain adducts clear associations were established. OHdG in urine and lymphocytes: not known.
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Not all endpoints are related to cancer risks !!!!

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Micronuclei are formed as a consequence of chromosome breaks and aneuploidy

Micronucleus containing an entire chromosome

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Historical notes
Micronuclei were discovered by Schmid in 1972. The first experiment with exfoliated cells was conducted by Stich and Parida in 1982. The first FA study with nasal cells was published in 1992 by Ballarin et al. A review on the effects of FA in exfoliated cells appeared in 2006 (Speit et al.) In 2004 the HUMN xl consortium was formed (M. Fenech, E. Zeiger, N. Holland, C. Bolognesi, S Burgaz, S. Knasmller) and started to evaluate and standardize MN experiments with exfoliated buccal cells.
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Methodological aspects- experiments with buccal cells

Holland et al., Mutat Res 2008

Cells migrate from the basal membrane to the surface and are replaced by new cells; during this process the cells divide and form MN.
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Different types of nucelar aberrations can be identified in the cells

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Some endpoints reflect cytotoxicity others are considered to be a consequence of genetic damage
Genotoxic effects: Micronuclei: aneugenic and clastogenic effect Binucleates: spindle disturbance Nuclear buds: gene amplification Cytotoxicity: Pycnosis Karyolysis Condensed chromatin Karyorexis
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Nat Protoc. 2009;4(6):825-37. 2009 Buccal micronucleus cytome assay. Thomas P, Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmueller S, Fenech M

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Development of a standardized protocol

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Important aspects

Staining Preparation of optimal slides with a cytospin centrifuge Number of cells that has to be scored Number of individuals

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Staining
A large number of earlier studies used DNA unspecific stains (e.g. Giemsa) which lead to false positive results (as they stain keratin bodies that are formed in epithelial cells as a consequence of cytotoxicity.

Figures a and c depict May-Grnwald-Giemsa stained cells. Figures b and d show the same cells after staining with Feulgen and DAPI. Nersesyan et al. Cancer Epidemiol Biomarkers Prev 2006;15(10). October 2006
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Slide preparation

Optimal slide preparation is achieved by use of a cytospin. Direct smears will lead to low quality slides that are hard to evaluate.

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Number of cells which should be scored


Ceppi et al. (2010) suggest to score per individual 4000 cells, this number is rarely reached in earlier studies.

However Thomas et al. 2009 suggest scoring of 2000 cells.


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Number of participants requested

The number of participants which should be evaluated depends on the magnitude of the effect. Ceppi et al. (2010) state that 120 individuals (60 controls and 60 exposed) have to be analyzed in order to detect a 50% increase of MN over the background.

Mutat Res. 2010 Jul-Sep;705(1):11-9. Ceppi M, Biasotti B, Fenech M, Bonassi S. 17


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Average background frequencies of different anomalies


Nuclear anomaly MNed cells Nuclear buds (broken egg) Binucleated cells Karyorrhectic cells Pycnotic cells MNed cells Number of subjects 703 789 MeanSE 0.830.04 1.360.08 Bonassi et al., 2011 852 408 210 Metanalysis of 63 studies 3.040.20 2.230.25 4.380.52 1.100.55 Ceppi et al., 2010 Ref.

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Methodological aspects: experiments with nasal cells


The morphological features of the anomalies are similar as those found in buccal cells; however, the nuclei of the cells are larger, many ciliated cells are found in the smears and also goblet cells as well as lymphocytes and granulocytes.

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Numbers in the picture A indicate: 1 - ciliated cell with 3 MNi 2 - nuclear bud 3 - normal ciliated cell 4 - karyorrhexis 5 - karyolysis 6 - condensed chromation Picture B - 2 normal ciliated cells

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Methodological aspects

The collection of cells with a cytobrush is more tricky as the collection of cells from the oral cavity. The site of collection was shown to have an impact on the results. Apart from nuclear anomalies also metaplastic and dysplastic cells can be scored.
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Figure 1. Nasal cytopathologic findings in nonsmoking customs officers exposed to DEE throughout the year (Papanicolaou stain). (A) Squamous cell metaplasia. (B) Cylinder cell dysplasia. (C) Squamous cell dysplasia with mitotic figure (arrow). The scale bar in (B) also applies to (A) and (C).

Glck et al. 2003 Environ Health Perspectives

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The current data base

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MN studies with exfoliated buccal cells

In total ca. 300 studies have been published so far. They concern the effects of lifestyle factors (betel chewing, smoking), health status, dietary factors and occupational exposure.

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MN studies with nasal cells


In total only 19 studies were published, in most of them only MN were scored (three studies included also other nuclear anomalies). The investigations concerned the effects of occupational exposure (n=17/ 8 FA exposure), 2 investigations concerned air pollution in cities.

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Studies conducted in Vienna (ICR)


Chicken manure Welders Khat chewers Coca chewers

B:

/N

B:

/N+

B: +

B:

Smoking

B: + (2 studies)
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ring trial (in progress)


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Which endpoints are the most sensitive ones???

Nersesyan et al. 2006 Smokers of unfiltered cigarets

Fold increase over background

Fold increase over background

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Fold increase over background

Celik et al. 2004 painters (n=60 in each group)

Rekhadevi et al. 2010 fuel filling station attendants (n=100 in each group)
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20

15

15

10

10

MN

BE

KR

MN

BE

MN

BE

KR

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Which cell types are more sensitive, nasal or buccal cells?


Gonsebatt et al. 2000 Air pollution in Mexico City (n=20/group)
0.4 0.3 0.2 0.1 0.0 1.6 1.4 1.2 1.0 0.8

Burgaz et al. 1998 Welders I (n=32/group)

MN/1000 cells

Nasal Buccal Nasal Buccal Control area Polluted area

MN/1000 cells

Nasal Buccal Nasal Buccal Controls Exposed Wultsch et al. unpublished Welders II (n=25/group)
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Titenko-Holland et al. 1996 formadehyde exposure (n=19/group)


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MN/1000 cells

MN/1000 cells

Nasal Buccal Nasal Buccal Controls Exposed


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Nasal Buccal Nasal Buccal Controls Exposed

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Why experiments with exfoliated cells? (comparison of advantages and disadvantages in relation of experiment with lymphocytes)
Lymphocytes Large database (ca 2100 studies) Automated evaluation available sampling invasive long turnover time of lymphocytes Efficient DNA repair Indirect relation to cancer Buccal/Nasal cells only around 300 studies automated evaluation in preparation sampling not invasive short turnover time (2-3-weeks) low repair capacity of the cells (higher sensitivity) 90% of cancers are of epithelial origin
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FA studies with exfoliated cells

Since 2006 four further studies were published


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Micronucleus frequencies in exfoliated buccal cells of human subjects after formaldehyde exposure
Study size (subjects/cells scored) Exposur e (ppm) 29 Pre- and post exposure 1500 cells/subject 28 Pre- and post exposure 5004000 cells/per subject 28 Exposed/18 controls 3000 cells/subject 80 Exposed/85 Controls 2000 cells/subject 16 Exposed/23 controls ????? cells/subject 56 Exposed/85 controls 2000 cells/subject 21 Pre- and post exposure 1000 2000 cells/subject 1.4 (peak: 6.6) 1.4 (peak: 6.6) 24 0.25 Result: buccal cells Ref. (exposed ()/controls ()) Feulgen/Fast green 0.6/0.046 Suruda et al., 1.6 1993 FISH/propidium iodide 2.0/0.6 3.3 Staining Notes

0 MN in 22 males, but 0.19 in females

TitenkoFirst study of buccal Holland et al., cells with FISH 1996 technique Burgaz et al., 2002 Viegas et al., 2010 -

Feulgen /fast green 7.1/3.3 2.2 Feulgen/no counter stain 1.27/0.13 9.8 0.64/0.13 4.9 0.86/0.57 0.96/0.16 6.0 1.33/0.90

0.1

Wright's stain

Ying et al., 1997

Not reliable results because of stain

0.16 13.5

Feulgen/no counter stain DAPI

Ladeira et al., 10.4/ 2.8 2011 Speit et al., 2007 Exposure under controlled conditions

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Micronucleus frequencies in exfoliated nasal cells of human subjects after formaldehyde exposure
Study size (subjects/cells scored) 15 Exposed/15 controls 6000 cells/subject 29 Pre- and post exposure 1500 cells/subject Exposure (ppm) 0.10.39 1.4 (peak: 6.6) Staining Result: nasal cells (exposed ()/controls ()) 0.90/0.25 3.6 0.5/0.41 Ref. Notes

Feulgen/Fast green

Ballarin et al., 1992 Suruda et al., 1993

Feulgen/Fast green

13% increase in males, 37% in females

28 Pre- and post exposure 5004000 cells/per subject

1.4 (peak: 6.6)

FISH/propidium iodide

2.5/2.0

Titenko-Holland First study of nasal et al., 1996 cells with FISH technique

23 Exposed/25 controls 3000 cells/subject 18 Exposed/23 controls 3000 cells/subject 16 Exposed/23 controls ????? cells/subject

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Feulgen/Fast green

1.0/0.61 1.6 2.70/1.25 2.2 3.9/1.2 3.3 0.21/0.17

Burgaz et al., 2001 Ye et al., 2005

Not reliable results because of stain Not reliable results because of stain Exposure under controlled conditions 33

Wright's stain

0.1

Wright's stain DAPI

Ying et al., 1997 Zeller et al., 2011

41 Exposed/the same subject were controls 1000-2000 cell subject 0 to 0.7

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Comments I

Two studies used Wright's stain (a modified Giemsa staining procedure), their results are irrelevant as unspecific stains give misleading results. In one study (Suruda et al. 1993) no MN were found in samples from males (controls), this result is out of the acceptable range calculated by Bonassi et al. (2011) and is therefore, irrelevant.

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Comments II
The results of the remaining studies are controversial; none of them fulfills the criteria for optimal investigations Ladeira et al (2011) and also Viegas et al (2010) conducted relatively large studies; they evaluated 2000 cells and used Feulgen without counter stain. All other studies were smaller as suggested and in some of them the number of evaluated cells was below 2000 (Holland et al, 1996, Zeller et al, 2011). In none of the FA studies published so far nuclear anomalies other than MN were evaluated.
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Overall conclusions
The methods for MN studies with exfoliated cells have been substantially improved in the last years by the joint efforts of the HUMN XL consortium. It was shown that the MN frequencies of exfoliated cells of the oral cavity correlate with human cancer risks and are therefore valuable biomarkers for the detection of exposure to genotoxic carcinogens. Nasal cells have been less frequently used as oral cells but the morphological characteristics of their nuclear anomalies are similar as those seen in buccal cells. Results obtained so far with FA exposed individuals yielded controversial results; three studies in which positive results were obtained are inadequate due to methodological shortcomings.

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