Inhibitory effects of pesticides on growth and respiration of cultured cells

PESTICIDE

BIOCHEMISTRY

Inhibitory MASANORI

AND

PHYSIOLOGY

Effects YOSHIDA,

Depurtment

10,

313- 321(1979)

of Pesticides on Growth Cultured Cells

MICHIHIRO

ONAKA,

TOSHIO

of Agriculturnl

Chemistry.

Kyoto

and Respiration

FUJITA,

AND

University.

K.wto

MINORLI 606.

of

NAk.4JIMA

Jnptrrl

Received February 28, 1978: accepted August 24, 1978 Effects of various drugs including pesticides on the growth and respiration of cultured cells were evaluated comparatively using cell lines derived from mosquito ovary and subcutaneous mouse tissues. The concentration producing 50% inhibition of cell growth. I,, (M), was determined for each of 42 drugs. Inhibitors of respiration and nucleic acid and protein biosyntheses such as rotenone, piericidin A,, actinomycin D, and puromycin had very high PI,,, values of approximately 8. Except for the compounds known to be uncouplers of oxidative phosphorylation, the drugs suppressed the respiration rate of the cultured cells to various degrees. The PI,, value (and the pEC,,,, 150% enhancement of the control, value for uncouplers) was determined for each compound. By examining the relation of PI,, (and pEC,,,) values between cell growth and respiration, the compounds could be classified into two groups according to their modes of inhibitory action against the cultured cells. One mode relates to the inhibition of energy synthesis and the other. perhaps, to interference with the biosynthesis of biomacromolecules.

evaluated. We here discuss whether there are essential differences between the toxicities against insect and mammalian cells, and the possible relationships between toxicity and the effect on the cell respiration.

INTRODUCTION

Cultured cells from various animal origins have been used to investigate the mechanism of the cytotoxic action of various environmental chemicals (1 - 10). Most studies, however, have attempted to relate the toxicities of chemicals against cell growth in terms of their inhibitory effects on the biosynthesis of DNA, RNA, and/or protein using a single cell line at a time. It is generally acknowledged that cytotoxicity is related to the inhibition of the biosynthesis of these biomacromolecules. Nevertheless, studies which allow direct comparisons between the toxicities against cell lines from entirely different origins are needed. Since the biosynthesis of biomacromolecules is regarded as being the end point of various cellular functions, some specific cellular functions and/or components should be of use as markers for cytotoxicity. We determined the potency to inhibit growth of 42 compounds, including a variety of pesticides, against cell lines derived from mosquito ovary and subcutaneous mouse tissues. Effects on cell respiration in each of the cultured cell lines have also been

MATERIALS

AND

METHODS

Conzpounds. The compounds listed in Table 1 were used. They are broadly classified as (1) compounds interfering with respiration, (2) those interfering with nucleic acid and protein biosyntheses, (3) alkylating agents and mutagens, (4) neurotoxicants, and (5) others. Most compounds were either purified by repeated recrystallization or were of the purest grade commercially available. Mefenamic and flufenamic acids were the gift of Professor H. Terada, Tokushima University. SUD. Sumithion, parathion, and SF6847 were provided by the Sumitomo Chemical Industry Company. Piericidin A, (11) and callicarpone (12) were supplied by Professors N. Takahashi, The University of Tokyo, and K. Kawazu, Okayama University, respectively . 313 0048-357517910303 13-09$02.00/O Copyright rD 1979 by Academic Pres\. Inc. All rights of reproduction in any form rrerved.

on the Growth

(0.17) (0.13) (0.12)

(0.12)b (0.11) (0.15) (0.16) (0.11) (0.20) (0.02) (0.13) (0.15) (0.13) (0.15) (0.14) (0.08) (0.23) (0.14) (0.15) (0.10) (0.09) (0.12) (0.11) (0.05) (0.10) (0.11)

Culex cell -

1

8.55 5.98 5.55

8.74 7.75 8.50 <3.37c 5.24 3.75 3.98 5.98 5.47 6.69 7.32 4.06 5.21 4.86 4.62 6.37 4.97 4.52 4.80 3.71 5.24 4.14 4.41

L-cell

(0.15) (0.18) (0.12)

(0.12) (0.05) (0.11)

(0.19)

(0.18) (0.15) (0.08) (0.24) (0.17) (0.17) (0.31) (0.09) (0.15) (0.16) (0.23) (0.18) (0.26) (0.03) (0.16)

(0.12) (0.15) (0.12)

and Respiration

pIso for growth (M-I)

Compounds

6.76 7.71 8.59 3.73 5.25 4.3 1 4.83 6.72 5.15 6.83 6.62 4.29 4.32 4.15 4.91 7.40 4.37 4.98 4.29 4.18 4.40 4.37 4.13

of Various

Inhibitors of protein and nucleic acid biosynthesis (24) Actinomycin D” 8.39 (25) CycloheximideO 5.41 (26) Puromycin 5.97

Compounds interfering with respiration (I) Piericidin A,” (2) Rotenone” (3) Antimycin A,” (4) Amytal” (5) Papaverine” (6) KCN (7) Phenazine” (8) Phenylmercuric acetate” (9) Captan (10) Tri-n-butyltin chloride” (II) Triphenyltin acetate” (12) Cellocidinn (13) Dichloned (14) Chloranild (15) 8-Hydroxyquinoline” (16) SF6847”~’ (17) 2,4-Dinitrophenol” (18) 2.4-Dinitro-a-naphthol” (19) Pentachlorphenol” (20) p-Nitrophenol” (21) 4,6Dinitro-o-cresol” (22) Flufenamic acid” (23) Mefenamic acid*

Effects

TABLE

<4.55? C4.05’ C4.13’

(0.23) (0.18)

(0.15) (0.20) (0.47) (0.15)

(0.33) (0.23)

(0.26) (0.40) (0.24)

<4.55c <4.05c C4.13’

4.13f 4.62/ 3.63f 4.54’ 4.14’ 3.58/

4.21’

5.99f

7.93 7.80 7.94 <3.37( 5.06 3.81 c3.65’ 5.20 5.24 5.86 5.54 C3.79’ 5.21 4.53 ~3.28~

pIm for respiration (M-l)

and L-Ceils

Culex cell

Cells

8.04 7.20 8.38 <3.37c 4.91 3.74 <3.6Sc 4.98 5.50 6.14 6.16 c3.27’ 5.44 4.29 c3.28’

of Culex

L-cell

(0.11) (0.21) (0.14) (0. IO) (0.04) (0.14) (0.18) (0.19)

(0.08) (0.04)

(0.15) (0.06) (0.35) (0.23)

(0.17) (0.11)

(0.21) (0.10) (0.20)

10.19)

3.73 5.37

4.32 4.28 4.30 4.29 4.83 4.29 4.33 4.02 13.94’

6.03

5.19

4.05

c3.36’ 5.70

L-cell

(0.05) (0.15)

<3.59’ <3.92’

3.90 4.00 4.14 4.12 3.95 3.97 3.89 <3.68’ <3.94’

C3.79’

(0.01) (0.16) (0.12) (0.06) (0.10) (0.10) (0.15) (0.08) (0.17)

<4.17’

<3.14’

C3.41’ <4.28’

(0.16) (0.06) wJ4 (0. IO) (0.13) (0.07) (0.1 I)

13.59’ C3.92”

3.71 3.92 3.74 3.88 4.05 13.84’ 3.99 (3.68’ C3.94”

<3.92’

C4.17’

<3.14’

C3.41’ <4.28’

PI,, for respiration (M-l) Culex cell

(0.13)

(0.04)

(0.27)

‘I EtOH is used for the stock solution. h The estimated error is given in the parentheses. VThe pI$,, value was not definitively determined because of no or only a very weak inhibitory effect ‘j Me,SO is the solvent for the stock solution. *’ SF6847 3,5-Di-t-butyl-4-hydroxybenzylidenemalonon~tr~le. ’ pEC,,,, value. V SUD O-Ethyl-0-4-cyanophenyl-phenylphosphonothiolate.

c3.68’ 5.33

(0.05)

3.79

Others (4/) Chlordimeform (42) Callicarpone”

(0.09)

4.86

(0.15) (0.04) (0.15) (0.09) (0. IO) (0.18) (0.15)

(0.12)

4.17

4.70 4.61 4.43 4.50 4.64 4.48 4.55 ~3.68’ C3.94’

(0.10)

13.36’ S.06

Neurotoxicants (32) Lindane” Orrl (33) Aldrin” “I rl (34) Dieldrin” Orrl (35) p,p’-DDT” Or” (36) Parathion” (37) Sumithion” (38) SUD”,” (39) Carbaryl” (40) Neostigmine iodide

Alkylating agents and mutagens (27) Ethyl methanesulfonate” (28) 4-Nitroquinolinel-oxide” OrB (29) p-Tosyl-N-methylN-nitrosoamide” (30) N-Methyl-N’-nitroN-nitrosoguanidine” (31) 6-Thioguanine”

Culex cell

pl,, for growth (M-l) L-cell

(0.11)

(0.07) (0.10) (0.06) (0.08) (0.20)

316

YOSHIDA

srudies. The cell lines used were derived from the ovarian tissues of Culex pipiens var. molestus (culex cell) and from the subcutaneous tissue of a mouse (L-cell). These cell lines were inoculated into a Leighton tube (18 x 18 x 75 mm3) containing either modified Kitamura medium (13), the composition of which is given in Table 2, for the culex cells or Eagle’s minimum essential medium (Nissui Seiyaku Co. Ltd., Tokyo) supplemented with 4% calf serum for the L-cells. The volume of the medium, usually 1.5 ml, was selected so that the number of cells per milliliter in each tube would be 2-6 x IO’. Cells were incubated in the stationary position for 2 days for the culex cells and for 1 day for the L-cells until a monolayer of cells was developed. The culture medium was then exchanged for the same volume of fresh medium containing various concentrations of each compound and the cells were further incubated. After 4 days of incubation for the culex cells and 2 days for the L-cells, the medium was removed. Preliminary experiments showed that the sensitivity of cell growth to serial dilution of the compounds is highest after the above incubation periods for each cell line. All operations were performed at 25°C TABLE

2

Composition of Modified Kitamura Medium” Compound NaCl KC1 CaCI, .2H,O KH,PO, NaHCO, Glucose Lactoalbumin hydrolysate Hz0 Medium 199 with Hanks’ salt solution Fetal calf serum Kanamycin sulfate

Amount 390 mg 30 mg 6 m3

6 mg 6 mg 120 mg 600 mg 60 ml 40 ml 5 ml 10 mg

” The pH of the medium was adjusted to 7.4 with KOH.

ET

AL.

for the culex cells and at 37°C for the L-cells. The monolayer of cells was gently scraped off using a silicon rubber policeman in 1.5 ml of phosphate buffer saline solution (NaCl, 8 g; Na2HP0,*2H20, 1.15 g; K,HPO,, 0.20 g; KCl, 0.20 g in 1000 ml of HzO). A uniform cell suspension was obtained by repeated pipettings after which 0.75 ml of aqueous 0.5% trypan blue solution was added to the suspension. The number of living cells which were not stained by the trypan blue treatment was counted with a Burker-Turk hematometer. Without addition of chemicals into the cultured medium, the number of stained cells was negligible. Water-soluble compounds were directly dissolved in the medium. Other compounds were dissolved in either ethanol or dimethylsulfoxide and diluted in the medium. The solvents used for the stock solution are shown in Table 1. The final concentration of the organic solvents in the medium did not exceed 0.5% (v/v) even at the highest concentration for water-insoluble compounds. At this concentration, no toxic effect was observed due to organic solvents alone. The I,,, concentration for each compound which produces the number of living cells at 50% of the control was determined from the dose -response curve plotted on log-probit paper. Examples are shown in Fig. 1. Effects on cell respiration. Each cell line was cultured for 1 day longer than for the toxicity studies under similar conditions with 50 ml of the culture medium in a 500-ml Roux bottle. At the end of the culture period, the medium was exchanged for about 50 ml of fresh medium. The cell monolayer was mechanically dispersed, after which the cell number in each suspension was adjusted to 3 - 10 x 10” cells/ml by the addition of a small volume of fresh medium. Five milliliters of the suspension was pipetted into a test tube [6($)mm x 105(ht)mm] into which various amounts of drug solution were added with a microliter syringe. The suspension was shaken gently

INHIBITORY

EFFECTS

OF

PESTICIDES

Dose(M) 0 Parathion

vs.

CVLTURED

vs. Culex

cells Dose(M)

1. Dose -response

curves

.for

LINES

317

RESULTS

10-6

10-7 0 PiericidinAl

CELL

control, as shown in Fig. 2. For these compounds, the I,,, concentration was determined from the “steep” phase of the dose -response plot. Compounds which are known to be uncouplers with oxidative phosphorylation enhanced rather than retarded the rate of respiration at low concentrations. The concentration which causes 150% enhancement of the control, ECIso, was determined. Examples are shown in Fig. 3.

L-cells

gg%

FIG. Rrou’th

AGAINST

Qf

inhibition

and covered with a layer of liquid paraffin. The rate of oxygen consumption in the suspension was followed for a period of from 15 to 60 min after the addition of compounds using an oxygen electrode, YSI Clark oxygen probe (Yellow Springs Instrument Co., Inc., Yellow Springs, Oh.). The rate was determined for each run at 37°C in terms of pm01 O,/min/3 x lo5 cells/ml for the L-ceils and pmol O,/min/ IO6 cells/ml for the culex cells. The I,, concentration which causes a 50% suppression of the respiration rate was estimated for each compound from the dose-response curve. Compounds such as piericidin Al, rotenone, and papaverine did not suppress the rate of respiration below 40% of the

Cytotoxicity. The pIso values for cell proliferation are listed in Table 1 as the averages of at least two replications. In general, compounds showing a high toxicity against both cell lines are those which interfere with respiration and biosynthesis. Piericidin A, (I), rotenone (2), antimycin A, (3), organo-tin compounds (10, II), and actinomycin D (24) are among the most toxic compounds, their PI,,, values ranging from 6 to 8. In contrast, alkylating agents (27, 29) and neurotoxicants (32-40) are not very toxic. The toxicity of a number of insecticides against cultured cell lines from the larvae of Antheraea eucalipti and Aedes aegypti was studied by Mitsuhashi and his co-workers (1, 2). They found that rotenone is very toxic against both insect cell lines. The toxicities of other insecticides such as lindane, aldrin, dieldrin, p,p’-DDT. and 0

PCP

Dose(M) 10-b

2

90 I\

‘b, \

E 9 ‘i; 20

a

‘\ 0

50

0

&y- Rotenone ------------.(-&-‘E 0 a

11

II 10-7

’ “I” 10-a

50 -

I

10-7

FIG. 2. Dose -response respiration of the L-cells.

curves

for

the inhibition

l(r6 0 SF6347

Dose(M)

of

FIG. 3. Dose -response couplers on the respiration

Dose(M)

curves for the effect of L-cells.

of un-

YOSHIDA

318

ET

AL.

carbaryl are much lower. The effects of or- itory effects. While they show high toxicity ganochlorine, organophosphorus, and car- against cell proliferation, the inhibitors of bamate insecticides, chlordimeform, and protein and nucleic acid biosyntheses, as captan on mammalian cell cultures such as well as the alkylating agents and mutagens, the mouse L, HeLa, and heteroploid human do not inhibit cell respiration very effecembryonic cells have also been studied by tively. Although the degree of inhibition is many workers (3 - 10). Although the origins much lower, neurotoxicants such as comof the cultured cell lines are different, the pounds 32-38 have definite inhibitory efpublished toxicity data seem to show a fects. trend similar to that of the present results. DISCUSSION Effect on respiration. The PI,,, values The PI,, values for the inhibition of for those compounds lowering the rate of growth of both culex cells and L-cells are respiration and the pEC,,, values for those plotted against each other in Fig. 4. The fact accelerating the rate of respiration are that the highly toxic compounds are found shown in Table 1 as the averages of at least among the respiratory chain inhibitors, untwo replications. As expected, compounds couplers, and inhibitors of protein and known to inhibit the electron transport sys- RNA biosyntheses shows that the respiratem in mitochondria (I -5) are highly inhibtion and /or the biosynthesis of macitory (except for amytal (4)), while those romolecules are quite essential to the proclassified as uncouplers (16 -23) accelerate liferation of cultured cells. Except for oxygen consumption. Of the fungicidal piericidin A, (I) and 6-thioguanine (3/), the compounds, phenylmercuric acetate (8>, PI,,, values are almost linearly related. This captan (9), and the organo-tin compounds indicates that the mechanism of the inhibit cell respiration but phenazine (7), cytotoxic effect for each compound is alcellocidin (12), and 8-hydroxyquinoline (1.5) most equivalent irrespective of the origin of have practically no or only very weak inhibthe cells.

r

I

4

I

I

6

I

a

I

~1% for Grcrwth of Culex cells ofpl, valuesfor FIG. 4. Relation number accompanying each plot

the growthofculex is the compound

andl-cells. number shown

in this and thefoffowingjigures, in Table I.

the

INHIBITORY

Ln

I 4

I

FIG. 5. Relation culev

and

t

OF

PESTICIDES

I

a

6 pISO

tbr’een

EFFECTS

fcf Ffespirafionof ofp15,

values

Culex for

cells

respiration

he-

L-cells.

In Fig. 5, the PI,, values for the respiration of both cell lines are plotted against each other for those compounds for which a definite inhibitory activity was determined. This figure also shows a linear relationship indicating that factors contributing to the inhibition of respiration are almost equivalent for the two cell lines. As shown in Table 1, “noninhibitive” compounds against one cell line are generally “noninhibitive” against the other. As shown in Figs. 6a and b, the PI,, values for growth are almost linearly related to those for respiration in each cell line for the

L,

a

1

I

I

4

6 pga

FIG. Relation

6. (a)

of

for

Relation pl,, ~~alrtes

I

I

pi,, bcm~een

values between growth grm’th and respiratory

CULTURED

CELL

LINES

319

respiratory inhibitors (I -3, 5, 6) and nerotoxicants (32 -38). Against the growth of culex cells, piericidin A, (I) is less inhibitory than expected from its PI,, value for respiration, while the reverse is the case for phenylmercuric acetate (8). Against the growth of the L-cells, however, the inhibitory activity of these compounds corresponds to that expected from the PI,,, (respiration) value. The origin of the behavior of compounds I and 8 is not clear. The situation with culex cells is apparently more complex than with L-cells. Further studies are needed in this area. Figures 6a and b indicate that neurotoxic insecticides, e.g., compounds 32 -38, behave as inhibitors against cell respiration. The fact that these classes of compounds inhibit mitochondrial electron transport, probably acting on the cytochrome systems, has been shown by Bergen (14) and Johnston (15). In Fig. 7, the PI,,, values for L-cell proliferation are plotted for uncouplers against the pEC,,, values for respiration. The two values are linearly related and the plot is almost superposable on the linear phase in Fig. 6b. This indicates that the effect of respiratory inhibitors in reducing the rate of oxygen consumption to 50% of the control is nearly equivalent to that of the uncou-

4

a

Growth

of

AGAINST

1;

’ 4

b and respirutrvy inhibition jiv

I

I

,

6 ~150 inhibifion L-cells.

for

I

i

a

Growth for

cu1e.y

ceils.

th)

320

YOSHIDA

6 c

,oG

2

t t‘

1 2

I 4

I

pI.jo FIG.

PEC,,~

7. Relation values for

I 6

for Growth

between gron~thpI,Oandrespiration uncouplers against L-cells.

plers which enhance the rate to 150%. The common consequence is probably the suppression of energy synthesis in the form of ATP to a similar extent. Since the inhibitors of biosynthesis (24-26), the alkylating agents, and mutagens (28, 30), without interfering with cell respiration, are highly inhibitory against cell proliferation, the compounds tested can be classified into two groups according to their modes of inhibitory action against the growth of cultured cells: compounds which inhibit energy synthesis and others which perhaps interfere with the biosynthesis of biomacromolecules. Actinomycin D is known to inhibit DNA-dependent RNA synthesis while puromycin and cycloheximide inhibit RNA-dependent protein synthesis. In fact, using primary cultured cells derived from a hamster fetus, Nebert and Gelboin (16) found that 4 x lO-‘M actinomycin D shows 90% inhibition of r3H] uridine incorporation into the RNA in the cell and 6 x 10e5M puromycin and 3.5 x 10-W cycloheximide lower the “C-labeled-protein-hydrolysate incorporation into cellular protein up to 5% of that incorporated in the control after a 30-min exposure. Similar concentration values for these compounds in inhibiting the incorporation of low-molecular-weight components were observed for HeLa cells

ET

AL.

by Myhr (3). He also found that the I,, concentrations of aldrin, p,p’-DDT, and carbaryl against the amino acid incorporation are around 5 x 10p4M (3). Although the origins of the cell lines differ, the order of inhibitory effects on DNA and protein biosyntheses given in the literature does not seem to markedly differ from that for the cytotoxicity determined in this work. The present study indicates that the main origin of the toxicity of cultured cells is due either to interference with respiration or to inhibition of macromolecular biosynthesis irrespective of the origin of the cells. For compounds whose mode of toxic action on the whole animal or insect body is attributed to interference with either respiration or macromolecular biosynthesis, cultured cell lines should provide a convenient tool for analyzing the toxicity effect on the cellular level. ACKNOWLEDGMENTS

We wish to express our sincere gratitude to Dr. Michio Himeno for his guidance in carrying the cell culture technique. We are also indebted to Drs. Nobutaka Takahashi, Hiroshi Terada. and Kazuyoshi Kawazu and to Sumitomo Chemical Industry Company and Nihonshinyaku Company for supplying us with several valuable compounds. This work was partly supported by a grant from the Japan Ministry of Education. REFERENCES

1. J. Mitsuhashi, T. D. C. Grace, and D. F. Waterhouse, Effects of insecticides on cultures of insect cells, Entomol. Exp. Appl. 13,327 (1970). 2. J. Mitsuhashi, T. C. D. Grace, and D. F. Waterhouse, Studies on the effects of rotenone on the growth of insect cells cultured in vitro, Enromol. Exp. Appl. 13, 467 (1970). 3. B. C. Myhr, A screen for pesticide toxicity to protein and RNA synthesis in HeLa cells, J. Agr. Food Chem. 21, 362 (1973). 4. R. A. Chung, I-LO Hung, and R. W. Brown, Studies of DNA, RNA and protein synthesis in HeLa S cells exposed to DDT and dieldrin. J. Agr. Food Chem. 15, 497 (1967). 5. H. H. North and R. E. Menzer, Inhibition of growth and esterase activity in mouse fibroblast cell cultures by several organophosphorus insecticides, J. Agr. Food Chem. 18, 797 (1970). 6. C. L. Litterst, E. R. Lichtenstein, and K. Kajiwara, Effects of insecticides on growth of HeLa cells, J. Agr. Food Chem. 17, 1199 (1969).

INHIBITORY

EFFECTS

OF

PESTICIDES

7. R. Sheinman and S. Yannai, Toxicity of dieldrin to primary cultures of rat fetal cells and human kidney cell line, Toricol. Appl. Pharmuco/. 30, 266 ( 1974). 8. C. L. Litterst and E. P. Lichtenstein, Effects and interactions of environmental chemicals on human cells in tissue culture, Arch. Environ. Health 22, 454 (1971). 9. M. Murakami and J. Fukami, Effects of chlorphenamidine and its metabolites on HeLa cells, Bull. Environ. Contam. Taxicol. 11, 184 (19741. 0. Cl. R. Gale, A. B. Smith, L. M. Atkins, E. M. Walker, Jr., and R. H. Gadsden, Pharmacology of captan: Biochemical effects with special reference to macromolecular synthesis, To.rico/. Appl. Pharmacol. 18, 426 (1971). 1. M. Jeng, C. Hall, F. L. Crane, N. Takahashi. S. Tamura, and K. Folkers, Inhibition of mitochondrial electron transport by piericidin A

AGAINST

12. 13. 14. 15.

16.

CITLTURED

CELL

LIh’ES

321

and related compounds, Biochemisrr~ 7, I3 I I (1968). K. Kawazu, M. Inaba. and T. Mitsui, Studies on fish-killing components of Callicarpo vontiiCOIIS. Agr. Bid Chem. 31, 494 ( 1967). S. Kitamura, The itr rGtro cultivation of tissues from the mosquito. Cu/expipirr~.s var. molestus, Robe J. Med. Sc,i. 11, 23 ( 19651. W. G. Bergen. The in r*itro effect of dieldrin on respiration of rat liver mitochondria. Prm .Soc. Esp. Bid. Med. 136, 732 (1971). C. D. Johnston. The in vitro effect of DDT and related compounds on the succinoxidase \ystern of rat heart, Arch. Biwhem. Bioph\,.t. 31, 375 (19511. D. W. Nebert and H. V. Gelboin. Substrateinducible microsomal arylhydroxylase in mammalian cell culture. .I. Bifd. Chc,uf. 243, 6250 (1968).