Use of nasal and buccal cells in human biomonitoring ... - Formacare

Use of nasal and buccal cells in human 

biomonitoring studies for the detection 

of cytotoxic and DNA-damaging 

siegfried.knasmueller@meduniwien.ac.at 

chemicals 

Siegfried Knasmüller, 

A. Nersesyan, M. Misik 

Cancer Research Institute, Innere Medizin I 

Medicine University of Vienna 

1


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 

2


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 

3

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. 


4

Not all endpoints are related to cancer risks !!!! 


5


Micronucleus 

containing an entire 

chromosome 

Micronuclei are 

formed as a 

consequence 

of chromosome 

breaks and 

aneuploidy 

6


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. Knasmüller) 

and started to evaluate and standardize MN experiments 

with exfoliated buccal cells. 

7

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. 


8

Different types of nucelar aberrations can be 

identified in the cells 


9

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 


10

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 


11

Development of a standardized protocol…… 


12

• Staining 


Important aspects 

• Preparation of optimal slides with a cytospin 

centrifuge 

• Number of cells that has to be scored 

• Number of individuals 

13


Staining 

a b 

c 

Figures a and c depict May-Grünwald-Giemsa stained cells. 

Figures b and d show the same cells after staining with Feulgen and 

DAPI. 

d 

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. 

Nersesyan et al. Cancer Epidemiol Biomarkers Prev 

2006;15(10). October 2006 

14


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. 

15

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. 


16

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

Average background frequencies of different anomalies 

Nuclear anomaly Number of subjects Mean±SE Ref. 

MNed cells 703 0.83±0.04 

Nuclear buds (broken 

egg) 


789 1.36±0.08 

Binucleated cells 852 3.04±0.20 

Karyorrhectic cells 408 2.23±0.25 

Pycnotic cells 210 4.38±0.52 

MNed cells Metanalysis of 63 

studies 

Bonassi et al., 2011 

1.10±0.55 Ceppi et al., 2010 

18

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. 


19

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 

A 


B 

20


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. 

21

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). 

Glück et al. 2003 Environ 

Health Perspectives 


22


The current data base 

23

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. 


24


25

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. 


26

Chicken manure 

B: ─ / N ─ 

Studies conducted in Vienna (ICR) 

Welders 

B: + (2 studies) 


Khat chewers Coca chewers 

B: ─ / N + B: + 

B: ─ 

Smoking 

ring trial (in progress) 

27

Fold increase over background 

25 

20 

15 

10 

5 

0 

Which endpoints are the most sensitive 

ones??? 

Nersesyan et al. 2006 - 

Smokers of unfiltered cigarets 

MN BE KR 



8 

6 

4 

2 

0 

Celik et al. 2004 - 

painters (n=60 in each group) 

MN BE 

Rekhadevi et al. 2010 - 

fuel filling station attendants (n=100 in each group) 


25 

20 

15 

10 

5 

0 

MN BE KR 

28

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

MN/1000 cells 


Gonsebatt et al. 2000 - 

Air pollution in Mexico City (n=20/group) 

0.4 

0.3 

0.2 

0.1 

0.0 

4 

3 

2 

1 

0 

Nasal Buccal Nasal Buccal 

Polluted area Control area 

Titenko-Holland et al. 1996 - 

formadehyde exposure (n=19/group) 


Exposed Controls 




1.6 

1.4 

1.2 

1.0 

0.8 

3 

2 

1 

0 

Burgaz et al. 1998 - 

Welders I (n=32/group) 



Wultsch et al. unpublished - 

Welders II (n=25/group) 



29

Why experiments with exfoliated cells? 

(comparison of advantages and disadvantages in 

relation of experiment with lymphocytes) 

Lymphocytes Buccal/Nasal cells 

Large database (ca 2100 studies) only around 300 studies 

Automated evaluation available automated evaluation in preparation 

sampling invasive sampling not invasive 

long turnover time of lymphocytes short turnover time (2-3-weeks) 

Efficient DNA repair low repair capacity of the cells 

(higher sensitivity) 

Indirect relation to cancer ≥90% of cancers are of epithelial origin 


30

FA studies with exfoliated cells 

• Since 2006 four further studies were 

published 


31

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 


500–4000 cells/per subject 

28 Exposed/18 controls 


80 Exposed/85 

Controls 



????? cells/subject 



21 Pre- and post exposure 1000 - 


1.4 

(peak: 

6.6) 

1.4 

(peak: 

6.6) 


Staining Result: buccal cells 

(exposed 

(‰)/controls (‰)) 

Feulgen/Fast green 0.6/0.046 

↑ 1.6 

FISH/propidium 

iodide 

2.0/0.6 

↑ 3.3 

2–4 Feulgen /fast green 7.1/3.3 

↑ 2.2 

0.25 Feulgen/no 

counter stain 

1.27/0.13 

↑ 9.8 

0.64/0.13 

↑ 4.9 

0.1 Wright's stain 0.86/0.57 

↔ 

0.16 Feulgen/no 

counter stain 

0.96/0.16 

↑ 6.0 

13.5 DAPI 1.33/0.90 

↔ 

Ref. Notes 

Suruda et al., 

1993 

Titenko- 

Holland et al., 

1996 

Burgaz et al., 

2002 

Viegas et al., 

2010 

Ying et al., 

1997 

Ladeira et al., 

2011 

Speit et al., 

2007 

0 MN in 22 males, 

but 0.19‰ in 

females 

First study of buccal 

cells with FISH 

technique 

- 

- 

Not reliable results 

because of stain 

♀↑ 10.4/♂↑ 2.8 

Exposure under 

controlled 

conditions 

32

Micronucleus frequencies in exfoliated nasal cells of human subjects after 

formaldehyde exposure 

Study size (subjects/cells 

scored) 






500–4000 cells/per subject 






????? cells/subject 

41 Exposed/the same subject were 

controls 1000-2000 cell subject 

Exposure 

(ppm) 


Staining 

0.1–0.39 Feulgen/Fast green 

1.4 (peak: 

6.6) 

1.4 (peak: 

6.6) 

Feulgen/Fast green 

FISH/propidium 

iodide 

2–4 Feulgen/Fast green 

1 Wright's stain 

0.1 Wright's stain 

0 to 0.7 

Result: nasal cells 

(exposed (‰)/controls 

(‰)) 

0.90/0.25 

↑ 3.6 

0.5/0.41 

↔ 

2.5/2.0 

↔ 

1.0/0.61 

↑ 1.6 

2.70/1.25 

↑ 2.2 

3.9/1.2 

↑ 3.3 

DAPI 0.21/0.17 

↔ 

Ref. Notes 

Ballarin et al., 

1992 

Suruda et al., 

1993 

Titenko-Holland 

et al., 1996 

Burgaz et al., 

2001 

Ye et al., 2005 

Ying et al., 1997 

Zeller et al., 

2011 

- 

13% increase in 

males, 37% in 

females 

First study of nasal 

cells with FISH 

technique 

- 





Exposure under 

controlled 

conditions 33


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. 

34


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. 

35


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. 

36

Use of nasal and buccal cells in human biomonitoring ... - Formacare

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