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Structural chromosomal aberration rates and sister-chromatid exchange frequencies in females with Type 2 (non-insulin-dependent) diabetes
117
Mutation Research, 143 (1985) 117-11 9 Elsevier
MRLet t. 0692
Structural chromosomal aberration rates and sister-chromatid exchange frequencies in females with Type 2 (non-insulin-dependent) diabetes Walter Vormittag Departm ent of Internal Medicine II, University of Vienna, and Ludwig Boltzmann Institute of Gerontolo gy, A-I090 Vienna (Austria) (Accepted 15 Februa ry 1985)
In vitro studies (Brown and Wu , 1977), testing with small animals (Renner and Miinzner, 1980) and chromosomal studies in patients with Type 2 diabetes (Watson et aI., 1976) suggest that at least so me of the sulphonylurea drugs ma y have a mutagenic effect. One should be cautious when interpreting the results of Watson et al. (1976) - particularly relevant to human studies, because the pretreatment aberration rates were not known and the diabetic state itself might have influenced the result s. Therefore, we decided to examine structural chromosomal aberrations and sisterchromatid exchange frequencies in peripherallymphocytcs of patients with Type 2 diabetes before starting treatment with sulphonylurea drugs. Materials and methods 20 females with Type 2 diabetes were investigated: structural chromosomal analysis was performed in 15 (aged 56-86 years, mean age 69.7 years) and sister-chromatid exchange frequency wa s determined in 10 probands (aged 54-84 years, mean age 68.6 years). In 5 patients both aberration rates and sister-chromatid exchanges were analysed. 9 probands had been known to be diabetics for so me time (I month-14 yea rs), and had been kept on a special diet, and in II probands diabetes had
been diagnosed just before cyto genetic analysis. Consequently, all patients could be treated adequately by diet plus sulphonylurea drugs. At the time of blood sampling for cytogenetic analysis all patient s had a fasting blood glucose level of at least 170 mg /IOO ml (9.4 mmoles/l) (range 170 mgOJo-341 mg OJo; 9.4-18 .9 mmoles/I). None of the patients had ever been exposed to known mutagenic drugs, isotope diagnostic or irradiation therapy. Most patients had not had a diagnostic X-ray or only a chest X-ray within the previous 5 years; one patient had undergone cholecystography 1 year previously, one patient had had an X-ray of the lumbar vertebral column 3 year s previously and one patient had had an Xray of the entire vertebral column I month prior to examination. Control values fo r different age groups of normal females were obtained using the same methods as for the diabetic probands of this study and ha ve already been published: two age groups (15-20 years and 76-85 years) of 15 females for stru ctur al chromosomal analysis (Vorrnittag , 1977) and three age groups (13-20, 30-55 and 75-84 year s) of 10 females for sister-chromatid exchanges (Vorrnittag, 1983); in this study the data of the old controlgroup were used for comparison . We have been working in this field for many
0165-7992/ 85/$ 03.30 © 1985 Elsevier Science Publishers B.V . (Biom edical Division)
11 8
TABLE I G RO UP VAL UES OF ST RUCT U RAL C H RO l\10S0 MA L A BE RRAT IONS O BSE RVE D IN DIAB ET ICS AN D CO NT RO LS G ro up
Mean age (years)
Dia betics 69.7
Num ber of Nu mber ( 070) of rnetap ha ses with ana lysed Gap Isogap Ch romat id C hro mo so mal metapha ses br eak break
1500
29
2
(1.93) (0 . 13)
Co ntro ls
79. 1
1500
30
5
(2 .00) (0.33)
C hro matid exchange
Dicent ric chromosome
A typical tra nslocatio n chro mo some
20
14
I
I
I
( 1.33)
(0 .93)
(0 .07)
(0.0 7)
(0 .07)
17
0
6
I
(0.4 0)
(0.07)
7 (0 .47)
year s, and have never ob served significant changes in no rmal structural aberration rat es in our laborat or y. It did not , there for e, a ppear to be necessar y to co nd uct con current co ntro ls. In our diagnostic routine work during th e time o f obtaining the diabetes material, no increased aberration frequencies were ob served . Structural chromosomal analysis. Peripheral lym ph ocytes were cultured fo r 48 h at 37°C (Moo rhead et al. , 1960): 20 ml of veno us blood was set up for sedimentatio n fo r 3-4 h at 4°C. 1- 2 ml of leukocyte-rich plasma was added to 8 ml Pa rke r' s medium 199 suppleme nted with 15070 calf seru m , penicillin and strepto myci n, and 0.2 ml ph ytoh em agglutinin (Well com e). Colcemid (Gibco), fin al concentration 0. 1 /lg/ml, was added 3 h before harvesting . The Giem sa-stained slides were coded fo r blind analysis. 100 metaphases per culture were exa mined ; all types of structur al aberrations were scored; isoch ro ma tid breaks, double acentric fragments and isochrornat id-gap breaks. were classified as 'chromosomal break s' (Table I). Metaphases with a break and ga pes) were tab ulated as met aphases with a break onl y (Table I) . Sister-chromatid exchange. Cultures were set up in the same way as describ ed ab ove but ma inta ined in the dark (lkushima and Wol ff , 1974) for 72 h in th e pre sence of 5-b romodeoxyuridine (Fluka ; final co ncent ratio n 3.0- 3. 3 /lg/ml). To dete ct sister-chromatid exchanges the ' fluo rescent plu s Giem sa technique' with Hoechst 33258 and subseq uent Giem sa stai ning (P erry and Wo lff, 1974) was used. 20-30 cells (in thei r second cycle)
( 1.13)
wit h well spread chromosom es were analysed per cultu re. Results
T able I shows the total aberr ation co unts for di ab etics and our old control gro up: the freq uencies o f cells with gaps , isogap s and chro mosomal bre ak s ar e ra ther similar in both gro ups . Our controls had more dicentric chro moso mes and the d iab etics had significantly mor e cells with chro ma tid break s (chr' = 6.32 , DF = I , P < 0.05); but it mu st be stated that th e chromatid bre ak frequencies of each indi vidual pa tient were within the norm al ra nge (0-4070, Vormittag, 1977). No corr elatio n between aberr ation rat es and the blood glucose level at the time o f withdrawal of blood for cytogenetic an alysis o r the (known) duration of diabetes could be de tected. P revious diagnostic X-rays had ob sviously not infl uenced the results, since patients with relatively high radiation expos ure (see Materials and Method s) sho wed no incre ased a berratio n rate s within th eir group . T he sister-chro matid exchange ra te (Tabl e 2) was higher in co ntrols (group mean = 7.0 ± 1.22) than in pati ent s (group mean = 6.0 2 ± 1.39) but the difference is not statistically significant (r-test) . Discussion
Watso n et al. (1976) ob ser ved increased freq uencies o f ga ps, isoga ps and chro moso me excha nge aberratio ns (dicentric chromosomes and tr ansloca-
119
TABLE 2 SIST E R-C H RO MAT ID EX CH A NG E FR EQ UE NCIES Donor
2 3 4 5 6 7 8 9 10 G rou p mean
Diabetics
Co nt ro ls
Age N
Mean value per cell
Ra nge
Age N
Me an Ran ge value per cell
84
82 78 75 84
8.6 5.7 5.9 6. 1 7. 1 9.4 6.7 6.2 6.9 7.8
63 79
25 30 30
6. 1 6.8 4.1
2- 13 2-15 1- 8
54 82 64 56 58 70 76
30 30 30 27 25 26 30
7.9 7.0 4.8 5.0 4 .2 7.4
2- /3 3-14 2- 9 1- 13 2-11 3-16 4-15
68.6
6 .9
6.02± 1.39
75 79 82 78 83 84
80.0
30 30 30 30 30 30 25 25 20 22
2-1 5 2- 13 2-10 2- 14 2-18 3-19 3-10 3- 13 2- 13 2-14
7.0 ± 1.22
N, number o f metaph ases an al ysed .
tions) but not of chromatid or chromosomal breaks in cultur ed lymphocytes of diabetics undergoing treatment with sulphon ylurea. The frequ encies of these aberration types are certainly not increased in our diabetes material, compared not only with the va lues of our controls for older peo ple but also the values found in young healthy females (Vormittag, 1977). The particularly high number of dicentrics in the control group may be related to the very great age of these probands . Therefore our resu lts seem to offer strong evidence that the findings of Watson et al. (1976) had actually been influenced by the antidiabetic drugs . The frequency of cells with chromatid breaks
was significantly higher (nP< 0.05) in the diabetic than in the control gro up. But the indi vidual values did not exceed the normal range (0-4 070) and with respect to po ssib le variation s of individual values, a significance at the 5070 level is prob abl y not sufficient to ensure a true difference. Sister-chromatid exchange frequencies found in diabetic s were within the normal range . According to thi s result, increased sister-chro matid exchange rates fo und in diabetics ha ve to be regarded as strong evidence for mutagenic exposure .
References Bro wn, R .F . , a nd Y. Wu (1977) Indu ct ion of siste r-chro ma tid exc ha nges in Chi nese ham ster cells by chlo rpropa mide, Muta tio n Res., 56 , 215-217. lkush ima , T ., and S. Wolff (1974) Sister- chromatid exc ha nges ind uced by light flashe s to 5-br om od eoxyur idine and 5-iododeo xyuri di ne substi tu ted C hinese ha mst er chro mo so mes, Exp . Cell Res ., 87, 15-1 9 . Moorhead , P .M. , I. e. Nowe ll, W.J . Mellman , D.M . Battips an d D.H . Hunge rfo rd (1960) Chromoso me preparat ion s of leuk ocyte s culture d fo rm peripheral blood , Exp . Cell Res ., 220 , 6 13-6 16. Pe rr y, P . , a nd S . Wolff (1974) New Gie msa meth od fo r the di ffe re ntial stai ning o f sister chro mat ids, Nat ur e (Lo ndo n), 25 1, 156-1 58. Ren ner, H .W ., and R . Munzner (1980) M utagenicity o f sulpho nylureas, Mutation Res., 77, 349-3 55. Vorrn itt ag, W. (1977) Chrom osom enunter suchungen bei Probandinnen zweier Altersgruppen , Ak t. Gero nto l. , 7, 23-33. Vo rrn itt ag, M . (1983) Effect of donor age o n sister chro ma tid excha nge frequency in cultured human lymphocytes, Akt. Ger ont ol ., 13, 79- 81. Watson, W .A .F ., J.e. Petrie, D .B . Galloway, I. Bullock and J .C . Gilbe rt (1976) In vivo cytogen eti c activity of sulpho nylurea dru gs in man , Mut a tio n Res. , 38, 7 1-8 0.
Mutation Research, 143 (1985) 117-11 9 Elsevier
MRLet t. 0692
Structural chromosomal aberration rates and sister-chromatid exchange frequencies in females with Type 2 (non-insulin-dependent) diabetes Walter Vormittag Departm ent of Internal Medicine II, University of Vienna, and Ludwig Boltzmann Institute of Gerontolo gy, A-I090 Vienna (Austria) (Accepted 15 Februa ry 1985)
In vitro studies (Brown and Wu , 1977), testing with small animals (Renner and Miinzner, 1980) and chromosomal studies in patients with Type 2 diabetes (Watson et aI., 1976) suggest that at least so me of the sulphonylurea drugs ma y have a mutagenic effect. One should be cautious when interpreting the results of Watson et al. (1976) - particularly relevant to human studies, because the pretreatment aberration rates were not known and the diabetic state itself might have influenced the result s. Therefore, we decided to examine structural chromosomal aberrations and sisterchromatid exchange frequencies in peripherallymphocytcs of patients with Type 2 diabetes before starting treatment with sulphonylurea drugs. Materials and methods 20 females with Type 2 diabetes were investigated: structural chromosomal analysis was performed in 15 (aged 56-86 years, mean age 69.7 years) and sister-chromatid exchange frequency wa s determined in 10 probands (aged 54-84 years, mean age 68.6 years). In 5 patients both aberration rates and sister-chromatid exchanges were analysed. 9 probands had been known to be diabetics for so me time (I month-14 yea rs), and had been kept on a special diet, and in II probands diabetes had
been diagnosed just before cyto genetic analysis. Consequently, all patients could be treated adequately by diet plus sulphonylurea drugs. At the time of blood sampling for cytogenetic analysis all patient s had a fasting blood glucose level of at least 170 mg /IOO ml (9.4 mmoles/l) (range 170 mgOJo-341 mg OJo; 9.4-18 .9 mmoles/I). None of the patients had ever been exposed to known mutagenic drugs, isotope diagnostic or irradiation therapy. Most patients had not had a diagnostic X-ray or only a chest X-ray within the previous 5 years; one patient had undergone cholecystography 1 year previously, one patient had had an X-ray of the lumbar vertebral column 3 year s previously and one patient had had an Xray of the entire vertebral column I month prior to examination. Control values fo r different age groups of normal females were obtained using the same methods as for the diabetic probands of this study and ha ve already been published: two age groups (15-20 years and 76-85 years) of 15 females for stru ctur al chromosomal analysis (Vorrnittag , 1977) and three age groups (13-20, 30-55 and 75-84 year s) of 10 females for sister-chromatid exchanges (Vorrnittag, 1983); in this study the data of the old controlgroup were used for comparison . We have been working in this field for many
0165-7992/ 85/$ 03.30 © 1985 Elsevier Science Publishers B.V . (Biom edical Division)
11 8
TABLE I G RO UP VAL UES OF ST RUCT U RAL C H RO l\10S0 MA L A BE RRAT IONS O BSE RVE D IN DIAB ET ICS AN D CO NT RO LS G ro up
Mean age (years)
Dia betics 69.7
Num ber of Nu mber ( 070) of rnetap ha ses with ana lysed Gap Isogap Ch romat id C hro mo so mal metapha ses br eak break
1500
29
2
(1.93) (0 . 13)
Co ntro ls
79. 1
1500
30
5
(2 .00) (0.33)
C hro matid exchange
Dicent ric chromosome
A typical tra nslocatio n chro mo some
20
14
I
I
I
( 1.33)
(0 .93)
(0 .07)
(0.0 7)
(0 .07)
17
0
6
I
(0.4 0)
(0.07)
7 (0 .47)
year s, and have never ob served significant changes in no rmal structural aberration rat es in our laborat or y. It did not , there for e, a ppear to be necessar y to co nd uct con current co ntro ls. In our diagnostic routine work during th e time o f obtaining the diabetes material, no increased aberration frequencies were ob served . Structural chromosomal analysis. Peripheral lym ph ocytes were cultured fo r 48 h at 37°C (Moo rhead et al. , 1960): 20 ml of veno us blood was set up for sedimentatio n fo r 3-4 h at 4°C. 1- 2 ml of leukocyte-rich plasma was added to 8 ml Pa rke r' s medium 199 suppleme nted with 15070 calf seru m , penicillin and strepto myci n, and 0.2 ml ph ytoh em agglutinin (Well com e). Colcemid (Gibco), fin al concentration 0. 1 /lg/ml, was added 3 h before harvesting . The Giem sa-stained slides were coded fo r blind analysis. 100 metaphases per culture were exa mined ; all types of structur al aberrations were scored; isoch ro ma tid breaks, double acentric fragments and isochrornat id-gap breaks. were classified as 'chromosomal break s' (Table I). Metaphases with a break and ga pes) were tab ulated as met aphases with a break onl y (Table I) . Sister-chromatid exchange. Cultures were set up in the same way as describ ed ab ove but ma inta ined in the dark (lkushima and Wol ff , 1974) for 72 h in th e pre sence of 5-b romodeoxyuridine (Fluka ; final co ncent ratio n 3.0- 3. 3 /lg/ml). To dete ct sister-chromatid exchanges the ' fluo rescent plu s Giem sa technique' with Hoechst 33258 and subseq uent Giem sa stai ning (P erry and Wo lff, 1974) was used. 20-30 cells (in thei r second cycle)
( 1.13)
wit h well spread chromosom es were analysed per cultu re. Results
T able I shows the total aberr ation co unts for di ab etics and our old control gro up: the freq uencies o f cells with gaps , isogap s and chro mosomal bre ak s ar e ra ther similar in both gro ups . Our controls had more dicentric chro moso mes and the d iab etics had significantly mor e cells with chro ma tid break s (chr' = 6.32 , DF = I , P < 0.05); but it mu st be stated that th e chromatid bre ak frequencies of each indi vidual pa tient were within the norm al ra nge (0-4070, Vormittag, 1977). No corr elatio n between aberr ation rat es and the blood glucose level at the time o f withdrawal of blood for cytogenetic an alysis o r the (known) duration of diabetes could be de tected. P revious diagnostic X-rays had ob sviously not infl uenced the results, since patients with relatively high radiation expos ure (see Materials and Method s) sho wed no incre ased a berratio n rate s within th eir group . T he sister-chro matid exchange ra te (Tabl e 2) was higher in co ntrols (group mean = 7.0 ± 1.22) than in pati ent s (group mean = 6.0 2 ± 1.39) but the difference is not statistically significant (r-test) . Discussion
Watso n et al. (1976) ob ser ved increased freq uencies o f ga ps, isoga ps and chro moso me excha nge aberratio ns (dicentric chromosomes and tr ansloca-
119
TABLE 2 SIST E R-C H RO MAT ID EX CH A NG E FR EQ UE NCIES Donor
2 3 4 5 6 7 8 9 10 G rou p mean
Diabetics
Co nt ro ls
Age N
Mean value per cell
Ra nge
Age N
Me an Ran ge value per cell
84
82 78 75 84
8.6 5.7 5.9 6. 1 7. 1 9.4 6.7 6.2 6.9 7.8
63 79
25 30 30
6. 1 6.8 4.1
2- 13 2-15 1- 8
54 82 64 56 58 70 76
30 30 30 27 25 26 30
7.9 7.0 4.8 5.0 4 .2 7.4
2- /3 3-14 2- 9 1- 13 2-11 3-16 4-15
68.6
6 .9
6.02± 1.39
75 79 82 78 83 84
80.0
30 30 30 30 30 30 25 25 20 22
2-1 5 2- 13 2-10 2- 14 2-18 3-19 3-10 3- 13 2- 13 2-14
7.0 ± 1.22
N, number o f metaph ases an al ysed .
tions) but not of chromatid or chromosomal breaks in cultur ed lymphocytes of diabetics undergoing treatment with sulphon ylurea. The frequ encies of these aberration types are certainly not increased in our diabetes material, compared not only with the va lues of our controls for older peo ple but also the values found in young healthy females (Vormittag, 1977). The particularly high number of dicentrics in the control group may be related to the very great age of these probands . Therefore our resu lts seem to offer strong evidence that the findings of Watson et al. (1976) had actually been influenced by the antidiabetic drugs . The frequency of cells with chromatid breaks
was significantly higher (nP< 0.05) in the diabetic than in the control gro up. But the indi vidual values did not exceed the normal range (0-4 070) and with respect to po ssib le variation s of individual values, a significance at the 5070 level is prob abl y not sufficient to ensure a true difference. Sister-chromatid exchange frequencies found in diabetic s were within the normal range . According to thi s result, increased sister-chro matid exchange rates fo und in diabetics ha ve to be regarded as strong evidence for mutagenic exposure .
References Bro wn, R .F . , a nd Y. Wu (1977) Indu ct ion of siste r-chro ma tid exc ha nges in Chi nese ham ster cells by chlo rpropa mide, Muta tio n Res., 56 , 215-217. lkush ima , T ., and S. Wolff (1974) Sister- chromatid exc ha nges ind uced by light flashe s to 5-br om od eoxyur idine and 5-iododeo xyuri di ne substi tu ted C hinese ha mst er chro mo so mes, Exp . Cell Res ., 87, 15-1 9 . Moorhead , P .M. , I. e. Nowe ll, W.J . Mellman , D.M . Battips an d D.H . Hunge rfo rd (1960) Chromoso me preparat ion s of leuk ocyte s culture d fo rm peripheral blood , Exp . Cell Res ., 220 , 6 13-6 16. Pe rr y, P . , a nd S . Wolff (1974) New Gie msa meth od fo r the di ffe re ntial stai ning o f sister chro mat ids, Nat ur e (Lo ndo n), 25 1, 156-1 58. Ren ner, H .W ., and R . Munzner (1980) M utagenicity o f sulpho nylureas, Mutation Res., 77, 349-3 55. Vorrn itt ag, W. (1977) Chrom osom enunter suchungen bei Probandinnen zweier Altersgruppen , Ak t. Gero nto l. , 7, 23-33. Vo rrn itt ag, M . (1983) Effect of donor age o n sister chro ma tid excha nge frequency in cultured human lymphocytes, Akt. Ger ont ol ., 13, 79- 81. Watson, W .A .F ., J.e. Petrie, D .B . Galloway, I. Bullock and J .C . Gilbe rt (1976) In vivo cytogen eti c activity of sulpho nylurea dru gs in man , Mut a tio n Res. , 38, 7 1-8 0.