Uptake and Persistence of Pesticides in Cultured Human Cells by MAKOTOMURAKAMIand JUN-ICHIFUKAMI Rikagaku Kenkyusho (The lnstitute oJ Physical and Chemical Research) IVako.shi, Saitama 351, ]apan

The uptake and persistence of a carcinogen, 3-methylcholanthrene in rat embryo cells have been reported ( ZIMMERMAN et al. 1975 ). However there have been few, if any, reports on the uptake and persistence of pesticides in cultured cells. As a p r e l i m i n a r y step to elucidate the m e c h a n i s m of pesticide toxicity at the cellular level, the incorporation of some pesticides into cultured human embryonic lung cells was studied. In the present study five insecticides were selected as test compounds. They were persistent chlorinated hydrocarbons, DDT, aldrin and dieldrin, an o r g a n o p h o s p h o r u s compound, parathion, and a novel b r o a d - s p e c t r u m acaricide-insecticide, chlordimeform (also known as chlorphenamidine or GalecronTM).

Materials and Methods

Human embryonic lung diploid cells ( HEL 299 ) ( PETERSON et al. 1968 ) from the A m e r i c a n Type Culture Collection (CCL 137) (Rockville, Maryland) were used in these experiments. The HEL cells were grown in Eagle minimal essential medium, supplemented with i0 % fetal bovine serum (Flow Laboratories, Rockville, Maryland) , sodium pyruvate (Ii0 mg/l), Bacto peptone (500 mg/l), glycine (7.5 mg/l), serine (10.5 mg/l), penicillin (50 units/ml), streptomycin (i00 ~ g / m l ) , and buffered w i t h Hepes buffer (5 mM) and N a H C O 3 (4.5 mM). Cells were grown as m o n o l a y e r cultures in tightly stoppered culture bottles at 37 ~ C. To determine the cellular uptake of pesticides,

cells

in 5 ml of growth m e d i u m were planted in 24 cm 2 culture bottles. After 24 hours of incubation the starter m e d i u m was replaced with 5 ml of m e d i u m and the cells were allowed to grow to confluence as indicated by microscopic examination. Radioactive pesticides (0.02)/mole) in 0.05 ml of ethanol were added to the cultures (Table i). ~ This dose of pesticides caused no visible c y t o t o x i c i t y to the cells. After a 24 hour incubation period this m e d i u m was replaced with 5 ml of fresh m e d i u m containing no test pesticide. At the time of this m e d i u m change, the cellular uptake of pesticide

425 Bulletin of Environmental Contamination & Toxicology, Vol. 15, No. 4 9 1976 by Springer-Verlag New York Inc.

TABLE Radioactive

Common name

Pesticides

1 Used

Chemical name

in This W o r k

Specific activity

Source

mCi/mmole

Chlordimeform

DDT

Aldrin a

N'-(2-Methyl-14C-4-chlorphenyl)-N,N-dimethylformamidine

34.5

l,l,l-Trichloro-2,2-bis(~-chlorophenyl-14C)ethane

6.25 and 5.88

1,2,3,4,10,10-Hexachloro-l,4,4a,

79

Parathion

a

Radiochemical Centre

1,2,3,4,10,10-Hexachloro-6,7epoxy-l,4,4a,5,6,7,8,Sa-octahydro1,4-endo-exo-5,8-dimethanonaphthalene

85

O,O-Diethyl-l-14C phosphorothioate

15.9

-O-s

New England Nuclear

5,8,Sa-hexahydro-l,4-endo-exo5,8-dimethanonaphthalene Dieldrin a

Schering

Radiochemical Centre

Radiochemical Centre

Carbon 1,2,3,4 and 10 positions are labeled with 14C.

at zero time was d e t e r m i n e d from two c u l t u r e bottles. The c u l t i v a t i o n was c o n t i n u e d for 9 or i0 days w i t h two a d d i t i o n a l c h a n g e s of m e d i u m d u r i n g the i n c u b a t i o n period. E a c h p o i n t (every two or three days) in the c e l l u l a r p e r s i s t e n c e c u r v e s is an a v e r a g e n u m b e r t a k e n from two bottles. The m e d i u m was r e m o v e d and the cell m o n o l a y e r s w e r e w a s h e d twice w i t h c a l c i u m and m a g n e s i u m free p h o s p h a t e b u f f e r e d saline. To the c u l t u r e s 2 ml of 1 % sodium d o d e c y l s u l f a t e (SDS) was a d d e d and m i x e d g e n t l y (HUBERMAN et al. 1971). Ten m i n u t e s later, 0.5 ml a l i q u o t s of the SDS s o l u t i o n w e r e p l a c e d into l i q u i d s c i n t i l l a t i o n c o u n t i n g vials. E t h a n o l (4 ml) was added, f o l l o w e d by i0 ml of the s c i n t i l l a t i o n m e d i u m p r e v i o u s l y d e s c r i b e d ( M U R A K A M I and F U K A M I 1974 ). The s a m p l e s were c o u n t e d for 5 m i n u t e s on a B e c k m a n L S - 1 5 0 L i q u i d S c i n t i l l a t i o n System. Counting e f f i c i e n c i e s w e r e d e t e r m i n e d by r e a d i n g d i r e c t l y f r o m the q u e n c h i n g c u r v e w h i c h was p l o t t e d a g a i n s t e x t e r n a l s t a n d a r d ratio, and c o u n t s per m i n u t e were c o n v e r t e d to d i s i n t e g r a tions per m i n u t e .

Results

and D i s c u s s i o n

C e l l u l a r u p t a k e and p e r s i s t e n c e of c h l o r d i m e f o r m , DDT and p a r a t h i o n are i l l u s t r a t e d in Fig. i. A t the b e g i n n i n g of the c u l t i v a t i o n w i t h the test i n s e c t i c i d e s , 426

DDT, a persistent chemical, was incorporated into the HEL cells about 20 to 30 times greater than the n o n - p e r s i s t e n t pesticides, chlordimeform and parathion. During incubation, the amounts of all pesticides taken up by the cells decreased w i t h time while m a i n t a i n i n g the initial proportions. In separate experiments, aldrin and dieldrin which are very stable in the environment gave cellular persistence curves identical w i t h that of DDT as shown in Fig. 2. Approximately i0 % of the p e r s i s t e n t insecticides added in the m e d i u m was initially incorporated into the cells. Based on the cellular uptake, the compounds tested clearly fall into two groups. One group comprises DDT, aldrin and dieldrin which are generally classed as persistent pesticides. During the initial stage of incubation the incorporation rates of these persistent compounds into the cells were approximately 20 to 70 times greater than c h l o r d i m e f o r m and parathion which are classified as n o n - p e r s i s t ent insecticides.

,~I 10!~

~ 102

OE lO

Chlordimeform

lO ?_ 4 6 8 Doys I

I

I

I

L

I0

Fig. i. Uptake and persistence of chlordimeform, DDT and parathion in cultured human embryonic lung cells.

Summary The uptake and persistence of 14C-labeled pesticides in cultures of human embryonic lung diploid cells were studied. Chlordimeform, DDT, parathion, aldrin and dieldrin were selected as test compounds. Results obtained from these experiments divided the chemicals into two groups. The first group consisted of three chemicals DDT, aldrin and dieldrin w h i c h are g e n e r a l l y classified as

427

P

persistent insecticides w h i l e the s e c o n d g r o u p was c o m p o s e d of c h l o r d i m e f o r m and p a r a t h i o n , g e n e r a l l y c o n s i d e r e d to be n o n - p e r s i s t e n t insecticides. The rate of i n i t i a l c e l l u l a r incorp o r a t i o n of f i r s t g r o u p was a p p r o x i m a t e l y 20 to 70 t i m e s g r e a t e r than the n o n - p e r s i s t e n t insecticides. During the c o u r s e of an i n c u b a tion, the a m o u n t s of p e s t i c i d e t a k e n up by the c e l l s d e c r e a s e d gradually.

10 4 -

\ G 10' o

E

"o

"~ 102 ci._

We w i s h to t h a n k Dr. K. F u k u n a g a for his i n t e r e s t and e n c o u r a g e ment. We a l s o t h a n k Dr. W. E. A l l i s o n , D o w C h e m i c a l Company, for his c r i t i c a l r e a d i n g of the m a n u s c r i p t .

100

I

0

I

4

i

I

6

8

I

10

Days Fig. 2. U p t a k e and p e r s i s t e n c e of a l d r i n and d i e l d r i n in c u l t u r e d h u m a n e m b r y o n i c lung cells.

References

H U B E R M A N , E., J . K . S E L K I R K , and C. H E I D E L B E R G E R C a n c e r R e s e a r c h 31, 2161 (1971). M U R A K A M I , M . , and J . F U K A M I l_!l, 184 (1974).

: Bull.

Environ.

:

Contam.

PETERSON,Jr.,W.D., C.S.STULBERG, N.K.SWANBORG, A . R . R O B I N S O N : Proc. Soc. Exp. Biol. Med. 128,

and 772

ZIMMERMAN,E.M., R.E.KOURI, K.HIGUCHI, F.LAIRD, A . E . F R E E M A N : C a n c e r R e s e a r c h 35, 139 (1975).

and

428

Toxicol

(1968).