Genotoxicity of the insecticide rotenone in cultured human lymphocytes

Mutation Research 414 Ž1998. 1–7

Genotoxicity of the insecticide rotenone in cultured human lymphocytes Ana Guadano Eduardo de la Pena ˜ ) , Azucena Gonzalez-Coloma, ´ ˜ Centro de Ciencias Medioambientales, CSIC, c r Serrano 115 dpdo, 28006 Madrid, Spain Received 28 November 1997; revised 10 February 1998; accepted 10 February 1998

Abstract We have investigated the genotoxic activity of rotenone on three genetic endpoints, sister-chromatid exchanges ŽSCE., chromosome aberrations ŽCA. and micronuclei ŽMN. in human lymphocyte cultures in the presence and absence of a metabolic activation system ŽS9 mix.. Our results indicate that rotenone increases the frequency of binucleated micronucleated ŽBNMN. cells and causes a delay in the cell cycle but does not increase the frequency of CA and SCE at the concentrations used. The presence of S9 mix reduces the genotoxic activity of rotenone. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Genotoxicity; Micronucleus; Rotenone; Lymphocyte; Natural insecticide

1. Introduction Rotenone is a natural insecticide isolated from the bark and root of plant species belonging to the genera Derris and Lonchorcarpus. Its specific inhibition of NADH-ubiquinone reductase has made it an important tool in the elucidation of the biochemical and molecular mechanisms of mitochondrial respiratory complex I. It has gained much attention as a reference for a new class of insecticidal compounds acting on the respiratory complex I as the primary biochemical target. The cytotoxicity of rotenone has been reported in cultured tumour cells as well as in solid human

) Corresponding author. Tel.: q34-1-5625020; fax: q34-15640800; E-mail: [email protected]

tumour types, although no cell-type specificity was found w1,2x. Additionally, carcinogenesis studies Ž2year feed studies. conducted in F-344rN rats and BGC3F1 mice gave unclear evidence of the carcinogenic activity of rotenone for male F-344rN rats and no evidence of carcinogenic activity in female F344rN and BGC3F1 mice w3x. The genotoxic activity of rotenone has been studied in different systems. Rotenone was not mutagenic when tested in a reverse mutation assay using Salmonella typhimurium w4,5x and in an unscheduled DNA synthesis assay using cultured human cells w6x. Rotenone induced forward mutations in the mouse L5178YrTK" lymphoma assay without activation w7x. Results of tests with rotenone in Chinese hamster ovary ŽCHO. cells were negative for induction of sister-chromatid exchanges ŽSCE. and chromosome aberrations ŽCA. w8x. However it induced endoredu-

1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 3 2 - 1

2

A. Guadano ˜ et al.r Mutation Research 414 (1998) 1–7

plication as well as aneuploidy and polyploidy in this cell line w9x. As part of our on-going research on the genotoxic activity of natural bioactive compounds acting on complex I w5x, the present study was designed to further investigate the genotoxic effects of rotenone. We have analysed its ability to induce different types of genetic damage in human lymphocyte cultures with and without metabolic activation by assessing three endpoints: CA, SCE and micronuclei ŽMN..

2. Materials and methods 2.1. Chemicals Rotenone ŽCAS no. 83-79-4., ethyl methanesulphonate ŽEMS, CAS no. 62-50-0., cyclophosphamide ŽCP; CAS no. 6055-19-2. and mytomicin C ŽMMC; CAS no. 50-07-7. were obtained from Sigma ŽSt. Louis, MO.. EMS was dissolved in bidistilled water. The other chemicals were dissolved in DMSO to give a final volume of solvent in the culture medium of 0.5% of the total. 2.2. Lymphocyte cultures Lymphocyte cultures were set up by adding heparinized whole blood to RPMI 1640 medium supplemented with 15% heat inactivated bovine serum, antibiotics Žpenicillin and streptomycin. and Lglutamine giving a final volume of 3 ml. Lymphocytes were stimulated by 2% phytohemagglutinin. For each genetic endpoint, blood from two different healthy non-smoking donors was used. 2.3. Metabolic actiÕation system The post-mitochondrial liver fraction supernatant ŽS9. from Wistar male rats weighing ; 200 g was used as the metabolic activation system. For induction we used a combined injection of PB and bnaphtoflavone w10x. The rats were injected i.p. with PB Ž30 mgrkg., PB Ž60 mgrkg., PB Ž60 mgrkg. and b-naphtoflavone Ž80 mgrkg. and PB Ž60 mgrkg., 4, 3, 2, and 1 dayŽs., respectively before they were sacrificed. The S9 liver fraction was prepared according to the method described by Maron

and Ames w11x. The protein concentration was 47 mgrml as determined by the Lowry et al. w12x procedure. Prior to the genotoxicity assays, the S9 mix was prepared by adding NADP and glucose-6phosphate Žboth obtained from Sigma. to S9. 2.4. Cytogenetic methods For the SCE study the cultures were incubated at 378C for 92 h. Twenty-four hours after the initiation of the cultures, 15 m grml of 5-bromodeoxyuridine ŽBrdU. were added and then the culture kept in the dark. After 20 h of incubation, the different concentrations of rotenone were added. Colcemid Ž0.2 m grml final concentration. was added 2 h prior to the termination of the culture. For CA, the cultures were incubated at 378C for 72 h, and the test compound was added 44 h after the initiation of the culture. Two hours prior to harvesting 0.2 m grml of colcemid were added. For the micronucleus assay the cultures were incubated as above. Forty-four hours after the initiation of the culture, the test compound was added together with 3 m grml of cytochalasin B to induce binucleated cells. The treatment with S9 mix in the three assays was short in duration Ž2 h. and performed in serum-free medium 44 h after the initiation of the culture. Following the treatment, the cells were washed twice with PBS and reconstituted in fresh tissue culture medium with serum. For each experiment, negative Žsolvent. and positive Žresponse to a standard mutagen. controls were included to ensure the validity of the assay. The cells were collected by centrifugation and resuspended in a hypotonic solution for 5 min at room temperature. We used a concentration of 0.06 M of KCl for SCE and CA and 0.01 M for MN since these concentrations were the best to obtain a good spreading of chromosomes and to preserve the cytoplasm, respectively wGuadano ˜ et al., unpublished resultsx. Following the hypotonic treatment, the cells were centrifuged and a methanol:acetic acid Ž3:1 vrv. solution was gently added. This fixation step was repeated twice. Two drops of a slightly turbid cell suspension were placed on a clean glass slide with a drop of acetic acid Ž40% vrv. for CA and SCE. The

A. Guadano ˜ et al.r Mutation Research 414 (1998) 1–7

preparations were air dried and the slides were then stained with 3% Giemsa in 0.1 M phosphate buffer pH 6.8 for 5 min ŽCA., 10% Giemsa for 15 min ŽMN. or with fluorescence plus Giemsa ŽSCE. w13x. 2.5. CA, SCE and MN analysis A hundred well spread metaphases were analysed for the CA assays, 50 second mitosis for the SCE assays and 1000 binucleated ŽBN. cells with preserved cytoplasm for MN, for each experimental concentration and donor on coded slides by a ‘blind’ study. In the CA studies, all the aberration data were recorded according to Savage’s description w14x. Gaps Žachromatid lesions. were scored to analyse the data excluding and including gaps. In addition, 1000 cells were analysed to obtain the mitotic index ŽMI. Žnumber of cells undergoing mitosisr1000 cells.. In the SCE study, 100 metaphases per point were also scored to obtain the Proliferative Rate Index ŽPRI., calculated according to the formula ŽM1 q 2M2 q 3M3r100., where M1, M2 and M3 represent the number of cells undergoing first, second, third and subsequent mitosis w15x.

3

In the case of the MN assay, 1000 BN cells per point were analysed to obtain the number of micronuclei Ž1, 2, ) 2. in BN cells and the number of BN micronucleated cells. A minimum of 500 lymphocytes were also scored to evaluate the percentage of BN cells with regard to total cells, i.e., mononucleated and BN cells. For the statistical analysis of the results, we used Fisher’s exact test for CA and micronucleated cells, the x 2 test for PRI and the t-test for SCE.

3. Results and discussion The rotenone concentrations evaluated ranged between 0.1 and 1 m grml. The highest dose was chosen by taking into account the level of cell toxicity found in previous experiments conducted in our laboratory. Tables 1 and 2 show the induction of chromosome structural aberrations for both donors treated with rotenone with and without metabolic activation. Cells exposed to several concentrations of rotenone, either in the presence or absence of S9 mix, did not show any significant increase in the frequency of CA

Table 1 Induction of chromosome structural aberrations in cultured human lymphocytes treated with rotenone—donor A Treatment

Concentration Ž m grml.

Chromatid-type aberrations G B E T

Chromosome-type aberrations Total aberrations G B E T yG qG

MI

48 h ŽyS9.

0 0.1 0.25 0.5 1 EMS Ž1.5 mM. 0 0.1 0.25 0.5 1 EMS Ž1.5 mM. 0 0.1 0.25 0.5 1 CP Ž3 m grml.

3 4 3 0 1 7 2 3 2 3 1 6 3 4 4 5 2 9

0 1 2 0 2 0 1 1 3 2 4 1 0 0 2 1 2 2

6.6 4.1 5.2 7.5 7.5 5.9 7.1 6.9 5.8 5.9 7.2 6.7 7.4 7.1 7.3 6.8 7.0 6.9

2 h ŽyS9.

2 h ŽqS9.

3 4 2 6 5 14 3 2 2 4 5 18 4 5 6 2 7 16

0 1 0 1 0 1 0 0 0 0 1 0 0 0 1 0 0 1

G, gaps; B, breaks; E, exchanges; T, total; MI, mitotic index. )) P F 0.01 ŽFisher’s exact test..

6 9 5 7 6 22 5 5 4 7 7 24 7 9 11 7 9 26

1 0 2 0 0 5 1 0 1 2 1 6 2 3 3 4 2 7

0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0

1 2 4 0 2 5 2 1 4 4 5 7 2 3 5 5 5 9

4 6 4 7 5

7 11 9 7 8

20 ) ) 4 2 3 6 7 24 ) ) 6 8 10 6 10 24 ) )

27 ) ) 7 6 8 11 12 31) ) 9 12 16 12 14 35 ) )

A. Guadano ˜ et al.r Mutation Research 414 (1998) 1–7

4

Table 2 Induction of chromosome structural aberrations in cultured human lymphocytes treated with rotenone—donor B Treatment

Concentration Ž m grml.

Chromatid-type aberrations G B E T

Chromosome-type aberrations Total aberrations G B E T yG qG

MI

48 h ŽyS9.

0 0.1 0.25 0.5 1 EMS Ž1.5 mM. 0 0.1 0.25 0.5 1 EMS Ž1.5 mM. 0 0.1 0.25 0.5 1 CP Ž3 m grml.

2 2 3 0 2 6 1 3 1 2 0 5 2 2 1 3 4 6

0 1 1 0 0 0 1 0 2 1 1 2 0 1 0 2 0 3

5.4 3.3 4.9 5.4 7.0 5.0 6.9 7.0 6.5 6.7 7.1 6.3 7.3 7.4 7.0 7.2 7.0 6.9

2 h ŽyS9.

2 h ŽqS9.

2 1 0 3 4 18 2 2 4 1 2 20 3 1 1 0 1 19

0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 1

4 3 3 3 7 24 3 5 5 3 2 26 5 3 2 3 5 26

2 2 3 1 1 4 1 1 1 3 3 2 3 3 3 2 2 5

0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0

2 3 4 1 1 5 2 1 3 4 4 4 3 4 3 4 2 8

4 3 3 4 6

6 6 7 4 8

23 ) ) 3 3 5 4 6 23 ) ) 6 4 4 2 3 25 ) )

29 ) ) 5 6 8 7 6 30 ) ) 8 7 5 7 7 34 ) )

G, gaps; B, breaks; E, exchanges; T, total; MI, mitotic index. )) P F 0.01 ŽFisher’s exact test..

for any donor, indicating a lack of insecticide clastogenicity under these conditions. These results are in agreement to those obtained in CHO cells w8x. The MI values also remained unaltered when compared to the control ones. The effects of rotenone on the SCE and PRI values are shown in Tables 3 and 4. The control values for SCE are very similar for both donors and they are not affected by rotenone at the concentrations tested. However, a dose response reduction of the PRI values was observed in the presence of rotenone suggesting a delay effect on the cell cycle. This reduction was very similar for both treatments Ž48 and 2 h. without S9 mix. In donor B, the effect of rotenone on PRI values in the presence of the S9 fraction, was only significant at the highest concentration used, indicating that the S9 fraction reduces that effect of rotenone in this donor. The results of the micronuclei study are shown in Tables 5 and 6. An increase in the frequency of BNMN cells was observed for the 48 and 2 h cultures treated with rotenone for both donors in the

Table 3 Induction of SCE and cell cycle delay by rotenone in cultured human lymphocytes—donor A Treatment

Concentration Ž m grml. SCErcell"SE PRI a

48 h ŽyS9. 0 0.1 0.25 0.5 1 MMC Ž0.2 m M. 2 h ŽyS9. 0 0.1 0.25 0.5 1 MMC Ž0.2 m M. 2 h ŽqS9. 0 0.1 0.25 0.5 1 CP Ž3 m grml.

5.14"0.86 4.94"1.11 5.51"1.06 5.37"1.18 4.82"1.09 36.63"2.14) 6.00"1.12 5.82"1.05 6.10"1.07 5.94"1.05 5.35"1.23 15.41"1.58) 7.35"1.36 6.91"1.41 7.04"1.35 7.41"1.44 7.69"1.31 21.06"2.47)

2.23 1.91) ) 1.95 ) ) 1.64 ) ) 1.40 ) ) 1.90 ) 2.14 1.85 ) 1.70 ) ) 1.44 ) ) 1.20 ) ) 1.97 2.17 2.09 ) ) 1.99 ) ) 1.85 1.70 ) ) 1.97 )

50 metaphases were scored for each concentration. a PRI, proliferation rate index. ) P F 0.05; ) ) P F 0.01 Ž t-test for SCE; x 2 test for PRI..

A. Guadano ˜ et al.r Mutation Research 414 (1998) 1–7 Table 4 Induction of SCE and cell cycle delay by rotenone in cultured human lymphocytes—donor B Treatment

Concentration Ž m grml. SCErcell"SE PRI a

48 h ŽyS9. 0 0.1 0.25 0.5 1 MMC Ž0.2 m M. 2 h ŽyS9. 0 0.1 0.25 0.5 1 MMC Ž0.2 m M. 2 h ŽqS9. 0 0.1 0.25 0.5 1 CP Ž3 m grml.

6.76"1.10 6.96"1.01 6.94"1.06 7.16"1.08 6.88"1.05 41.86"2.22 ) 7.05"1.85 6.98"1.51 7.10"1.41 6.57"1.14 7.28"1.31 22.12"2.69 ) 7.77"1.73 6.41"1.59 7.10"1.79 6.77"1.70 6.80"1.51 26.66"1.45 )

2.55 2.15 1.78 ) ) 1.79 ) ) 1.45 ) ) 1.93 ) 2.26 1.95 1.77 ) ) 1.45 ) ) 1.38 ) ) 2.01 2.31 2.30 2.04 1.98 1.85) 2.30)

50 metaphases were scored for each concentration. a PRI, proliferation rate index. ) P F 0.05; ) ) P F 0.01 Ž t-test for SCE; x 2 test for PRI..

5

absence of S9 mix, being this effect significant from concentration 0.25 m grml. The addition of the S9 fraction decreased the effect of rotenone. A dose-response decrease in the percentage of BN cells was also observed in the presence of rotenone. The genotoxic effects of rotenone have been investigated in various short-term assays with different results. The present study indicates that rotenone has no clastogenic effect and does not modify the frequencies of SCE in cultured human lymphocytes in the presence or absence of S9 mix. These observations are similar to those obtained in CHO cells. From our results it is also clear that rotenone induces a dose-response increase in the frequency of BNMN cells. To our knowledge this is the first time that the effect of rotenone on the frequency of micronuclei has been investigated. In the presence of rotenone there is a dose-response decrease of the PRI values and the percentage of BN cells, which seems to indicate a cytotoxic effect of the insecticide. However, if this were the case it should be accompanied by a parallel decrease

Table 5 Induction of micronuclei in human lymphocyte cultures treated with rotenone—donor A Treatment

Concentration Ž m grml.

BN cells Ž%.

BN scored

48 h ŽyS9.

0 0.1 0.25 0.5 1 MMC Ž0.2 m M. 0 0.1 0.25 0.5 1 EMS Ž15 mM. 0 0.1 0.25 0.5 1 CP Ž0,1 mM.

46.8 41.1 30.1 23.7 16.2 45.9 45.2 45.0 38.6 26.4 19.6 37.2 44.0 41.4 39.2 35.2 30.0 39.0

1000 1000 1000 920 900 1000 1000 1000 1000 950 900 1000 1000 1000 1000 1000 1000 1000

2 h ŽyS9.

2 h ŽqS9.

BN, binucleated; MN, micronuclei; BNMN, binucleated micronucleated cells. ) P F 0.05; ) ) P F 0.01 ŽFisher’s exact test..

MN in BN

Total MN

1

2

)2

2 5 9 12 16 35 4 7 10 16 19 54 5 6 8 10 12 38

0 0 1 2 0 3 0 0 1 0 1 12 0 0 1 0 0 2

0 0 1 0 2 0 0 0 0 0 0 2 0 0 0 0 0 1

2 5 14 16 22 41 4 7 12 16 21 84 5 6 10 10 12 45

Total BNMN

2 5 11) 14 ) ) 18 ) ) 38 ) ) 4 7 11) 16 ) ) 20 ) ) 68 ) ) 5 6 9 10 12 41) )

A. Guadano ˜ et al.r Mutation Research 414 (1998) 1–7

6

in the MI values. The lack of reduction of the MI values of the rotenone-treated cells could be explained by a metaphase-arresting effect. This effect would lead to an increase of first metaphase cells along with a delay in the cell cycle that, in turn, would affect the PRI values and the percentage of BN cells. The ability of rotenone to arrest cells in metaphase has been previously described w16,2x. These effects, along with the increased frequencies of BNMN cells observed in the lymphocytes treated with rotenone, suggest that this compound could act on the microtubule synthesis and assembly andror on the formation of the mitotic dividing spindle. Interference with such targets could lead to either chromosome loss, resulting from lagging chromosomes at the anaphase or chromosome non-disjunction where both sister chromatids migrate to the same daughter nucleus. The fact that rotenone does not induce enough chromosome or chromatid breaks as compared to control levels to explain the formation of micronuclei, points to the possibility that the observed increased frequency of BNMN cells could be a consequence of chromosome loss during mitosis. Interestingly, the presence of a metabolic activa-

tion system reduces the effect of rotenone on PRI values and BNMN frequency. Further studies are underway to investigate the effect of rotenone in the spindle apparatus and to check whether this effect is related to its action as a respiratory inhibitor.

Acknowledgements This work was supported by a grant from the DGICYT-Spain ŽPB 94rOO20rB.. We gratefully acknowledge Mr. S. Carlin for language revision and Dra. P. Gasco from the Spanish Institute of Toxicology for providing us with the facilities for S9 purification. We also thank Mrs. Antonia Martinez for help with the preparation of the manuscript.

References w1x G. Blasko, ¨ H.-L. Shieh, J.M. Pezzuto, G.A. Cordell, 13 Cy NMR spectral assignment and evaluation of the cytotoxic potential of rotenone, J. Nat. Prod. 52 Ž1989. 1363–1366.

Table 6 Induction of micronuclei in human lymphocyte cultures treated with rotenone-donor B Treatment

Concentration Ž m grml.

BN cells Ž%.

BN scored

48 h ŽyS9.

0 0.1 0.25 0.5 1 MMC Ž0.2 m M. 0 0.1 0.25 0.5 1 EMS Ž15 mM. 0 0.1 0.25 0.5 1 CP Ž0,1 mM.

52.7 47.7 34.9 22.1 19.8 49.4 49.4 43.2 35.6 28.0 20.6 36.4 50.0 47.2 40.2 35.0 33.4 45.2

1000 1000 1036 1000 920 1000 1000 1000 1000 1000 950 1000 1000 1000 1000 1000 1000 1000

2 h ŽyS9.

2 h ŽqS9.

BN, binucleated; MN, micronuclei; BNMN, binucleated micronucleated cells. ) P F 0.05; ) ) P F 0.01 ŽFisher’s exact test..

MN in BN

Total MN

1

2

)2

3 4 10 18 20 40 2 6 9 15 23 60 4 5 7 8 10 48

0 0 2 0 2 1 0 0 0 2 1 15 0 0 0 1 2 3

0 0 2 2 1 0 0 0 0 0 0 0 0 0 0 0 0 1

3 4 20 24 27 42 2 6 9 19 25 90 4 5 7 10 14 57

Total BNMN

3 4 14 ) 20 ) ) 23 ) ) 41) ) 2 6 9) )) 17 24 ) ) 75 ) ) 4 5 7 9 12 ) 52 ) )

A. Guadano ˜ et al.r Mutation Research 414 (1998) 1–7 w2x M. Loffler, F. Schneider, Further characterization of the ¨ growth inhibitory effect of rotenone on in vitro cultured Ehrlich ascites tumour cells, Mol. Cell. Biol. 48 Ž1982. 77–90. w3x K.M. Abdo, S.L. Eustis, J. Haseman, J.E. Huff, A. Peters, R. Persing, Toxicity and carcinogenicity of rotenone given in the feed to F344rN rats and B6C3F1 mice for up to two years, Drug. Chem. Toxicol. 11 Ž1988. 225–235. w4x M. Moriya, T. Ohta, K. Watanabe, T. Miyazawa, K. Kato, Y. Shirasu, Further mutagenicity studies on pesticides in bacterial reversion assay systems, Mutat. Res. 116 Ž1983. 185–216. w5x A. Guadano, C. Gutierrez, E. de la ˜ A. Gonzalez-Coloma, ´ ´ Pena, ˜ Mutagenic evaluation of two bioactive annonaceous acetogenins in Salmonella typhimurium strains TA98, TA100 and TA102, 1997, submitted for publication ŽMutation Res... w6x F.E. Ahmed, R.W. Hart, N.J. Lewis, Pesticide induced DNA damage and its repair in cultured human cells, Mutat. Res. 42 Ž1977. 161–174. w7x D.B. McGregor, A. Brown, P. Cattanach, I. Edwards, D. McBride, C. Riach, W.J. Caspary, Responses of the L5178Y tk"tk-Mouse lymphoma cell forward mutation assay: 72 coded chemicals, Environ. Mol. Mutagen. 12 Ž1988. 85–154. w8x B.E. Anderson, E. Zeiger, M.D. Shelby, M.A. Resnick, D.K. Gulati, J.L. Ivett, K.S. Loveday, Chromosome aberration and sister chromatid exchange test results with 42 chemicals, Environ. Mol. Mutagen. 16 Ž1990. 55–137.

7

w9x K. Matsumoto, T. Ohta, Rotenone induces aneuploidy, polyploidy and endoreduplication in cultured Chinese hamster cells, Mutat. Res. 263 Ž1991. 173–177. w10x T. Matsushima, M. Sawamura, K. Hara, T. Sugimura, A safe substitute for polychlorinated biphenyls as an inducer of metabolic activation system, in: F.J. de Serres ŽEd.., In Vitro Metabolic Activation in Mutagenesis Testing, ElsevierrNorth Holland Biomedical Press, Amsterdam, 1976, pp. 85–88. w11x D.M. Maron, B.N. Ames, Revised methods of Salmonella mutagenicity test, Mutat. Res. 113 Ž1983. 173–215. w12x O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. w13x P. Perry, S. Wolf, New Giemsa method for the differential staining of sister chromatids, Nature 251 Ž1974. 156–158. w14x J.R.K. Savage, Classification and relationships of induced chromosomal structural changes, J. Med. Genet. 12 Ž1975. 103–122. w15x L. Lamberti, P. Bigatti Ponzetto, G. Ardito, Cell kinetics and sister chromatid exchange frequency in human lymphocytes, Mutat. Res. 120 Ž1983. 193–199. w16x H.M. Meisner, L. Sorensen, Metaphase arrest of Chinese hamster cells with rotenone, Exp. Cell. Res. 42 Ž1966. 291–295.