Metal Carcinogenesis in Tissue Culture Systems - American Chemical

3 Metal Carcinogenesis in Tissue Culture Systems M A X COSTA Division of Toxicology, University of Texas Medical School at Houston, P.O. Box 20708, Houston, T X 77025 MARCIA K. JONES and ORRIN LINDBERG

Downloaded by PURDUE UNIV on April 9, 2016 | http://pubs.acs.org Publication Date: December 22, 1980 | doi: 10.1021/bk-1980-0140.ch003

Department of Pharmacology and Toxicology, College of Medicine, Texas A&M University, College Station, T X 77843

Metals present a human c a r c i n o g e n i c hazard p r i m a r i l y by i n h a l a t i o n , and t h e r e f o r e , induce primary neoplasms o f the r e s p i ratory t r a c t . A primary c o n s i d e r a t i o n i n metal carcinogenesis i s the p a r t i c l e s i z e o f the m a t e r i a l , since i t must be r e l a t i v e l y small (< 2 microns) to penetrate i n t o the a l v e o l a r spaces and induce lung cancer. A second important c o n s i d e r a t i o n i s the i d e n t i t y o f the s p e c i f i c metal compound because not a l l compounds of the same metal have s i m i l a r c a r c i n o g e n i c a c t i v i t i e s . A case in point i s the observation that c r y s t a l l i n e Ni3S? i s a very potent carcinogen i n experimental i s t e r e d by various routes to experimental animals, a large proportion o f these animals developed tumors. In c o n t r a s t , s i m i l a r animal exposure to amorphous NiS r e s u l t e d i n no n e o p l a s i a (_1 2). A d d i t i o n a l important c o n s i d e r a t i o n s i n c l u d e : 1) water o r l i p i d s o l u b i l i t y p r o p e r t i e s o f the metal c a r c i n o g e n , 2) dosage and p r e d i s p o s i t i o n o f the i n d i v i d u a l toward the development o f cancer. The l a t t e r point i s p a r t i c u l a r l y important i n l i g h t o f some o f the p r e l i m i n a r y r e s u l t s presented i n the experimental s e c t i o n o f t h i s paper showing that pretreatment o f c e l l s with benzopyrene p o t e n t i a t e s the carcinogenesis o f Ni'3S . Benzopyrene (found i n c i g a r e t t e smoke) and other x e n o b i o t i c s to which we are d i f f e r e n t i a l l y exposed t o i n our environment may g r e a t l y a l t e r the course of metal induced c a r c i n o g e n e s i s . In t h i s chapter we wish t o address ourselves s p e c i f i c a l l y to t i s s u e c u l t u r e systems f o r assaying metal c a r c i n o g e n i c a c t i v i t y . The r a t i o n a l e f o r t h i s i s the same as that f o r developing s i m p l e , r a p i d , r e l i a b l e , and inexpensive i n v i t r o cancer t e s t s f o r organic compounds. It i s impossible i n terms o f time and cost t o e v a l u ate hazards o f a l l the p o t e n t i a l l y c a r c i n o g e n i c metal compounds using the conventional animal carcinogenesis t e s t i n g systems. However, a s i n g l e i n v i t r o t e s t system i s probably i n s u f f i c i e n t in a s s i g n i n g c a r c i n o g e n i c a c t i v i t y to a p a r t i c u l a r compound. 5J

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0-8412-0588-4/80/47-140-045$07.25/0 © 1980 American Chemical Society Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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CHEMISTRY IN BIOLOGY A N D MEDICINE


A d d i t i o n a l l y , i n v i t r o t e s t s should not elimate animal t e s t i n g but may help i n t e s t s with animals. The proper use o f several in v i t r o t e s t systems w i l l g r e a t l y a i d our understanding of which metal compounds are hazardous. These screening t e s t s may a l s o allow us to i d e n t i f y carcinogens i n a i r samples from the i n d u s t r i al environment. Metals Have Caused Cancer i n Humans. Numerous recent reviews have been compiled which describe the various epidemiological studies i m p l i c a t i n g a r s e n i c , cadmium, chromium, and n i c k e l as causes of human cancer (3-11). These studies have analyzed the i n d u s t r i a l worker's exposure to these metals, or have examined s e l e c t e d populations having excessive exposure to these metals i n t h e i r d r i n k i n g water, the atmosphere adjacent to t h e i r h a b i t a t i o n , etc. The reader i s referred to these reviewers f o r a more comprehensive d i s c u s s i o n of the a v a i l a b l e data p e r t a i n i n g to metal carcinogenesis (3.-1J.). It should, however, be pointed out that n i c k e l and i t s compounds have been the most e x t e n s i v e l y documented c l a s s of metal carcinogens i n humans. There are several other metals, i n c l u d i n g b e r y l l i u m , which have also been l i n k e d to human cancer, but the most important metal carcinogens have been l i s t e d above (11). Metals Induce Cancer When Administered to Experimental Animals. Again, the reader i s r e f e r r e d to many recent comprehens i v e reviews which d i s c u s s the evidence that c e r t a i n metals cause cancer i n experimental animals ( 9 , ^ 0 , J 1 ) . The most c r e d i b l e data i m p l i c a t e s cadmium, chromium, co"5alt, and n i c k e l as carcinogens i n experimental animals. It should be noted that while a r s e n i c has been shown to be responsible f o r the i n d u c t i o n of human canc e r , attempts to induce cancer i n experimental animals with a r s e n i c and i t s compounds have not been s u c c e s s f u l . In c o n t r a s t , while cobalt induced cancer i n experimental animals, numerous epidemiological studies have f a i l e d to show a c o r r e l a t i o n between excessive human exposure to c o b a l t and the i n d u c t i o n of human neoplasia. One of the most studied metal carcinogens are the n i c k e l compounds, of which c r y s t a l l i n e Ni3S2 appears to be the most potent ( 1 , 2 , 1 2 ) . N i S has been shown to induce cancer at the s i t e of a d m i n i s t r a t i o n , r e s u l t i n g i n muscle tumors i f given by intramuscular i n j e c t i o n (I), renal tumors i f administered by i n t r a r e n a l i n j e c t i o n ( 2 j , t e s t i c u l a r tumors i f i n j e c t e d i n t o the t e s t e s ( 2 J , o r lung tumors i f administered by i n h a l a t i o n (12). In v i t r o A n a l y s i s of Metal Carcinogenesis. Methods of exp l o r i n g metal carcinogenesis w i l l be grouped i n t o three separate sections. The f i r s t group, biochemical s t u d i e s , includes those studies that deal with the basic i n t e r a c t i o n s of i s o l a t e d enzyme systems extracted from l i v i n g c e l l s . The second group considers the e f f e c t of metals on b a c t e r i a l systems, and the t h i r d reviews present techniques using t i s s u e c u l t u r e of c e l l s i n assessing metal c a r c i n o g e n e s i s . 3

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Studies

Most o f the studies that have examined the i n t e r a c t i o n s of metals i n biochemical systems have attempted to prove the hypot h e s i s that c a r c i n o g e n i c metals induce a l t e r a t i o n s i n the base p a i r i n g and f i d e l i t y of mRNA t r a n s c r i p t i o n i n v i t r o . These studies have suggested that a p r i n c i p a l mechanism o f metal c a r c i n ogenesis i n v o l v e s d i s t o r t i o n s i n t h i s t r a n s c r i p t i o n a l processes. One such i n v i t r o study examined the i n t e r a c t i o n between complementary and uncomplementary strands of s y n t h e t i c p o l y n u c l e o t i d e s which were mixed together and allowed to anneal (13). When synt h e t i c strands o f poly C-U (polycytosine monophosphate-polyuridine monophosphate) or poly C-A ( p o l y c y t o s i n e monophosphate-polyadenosine monophosphate) are mixed with the purine p o l y n u c l e o t i d e poly I (polyinosine monophosphate) i n the absence of any d i v a l e n t metal i o n , the strands h y b r i d i z e d because the cytosine residues formed hydrogen bonds with t h e i r complementary base, the hypoxanthine residues of the i n o s i n e (13_). If magnesium (2+) ions or n i c k e l (2+) ions are added to t h i s system, the degree o f h y b r i d i z a t i o n was i n c r e a s e d , suggesting the occurrence of uncomplementary base p a i r i n g . Normally, the u r a c i l and adenine n u c l e o t i d e s should not p a i r with the poly I, but i n the presence of magnesium or n i c k e l , the m i s p a i r i n g occurred (13). Cadmium has a s i m i l a r a f f e c t on the h y b r i d i z a t i o n o f poly C-A or poly C-U with poly I (1£). Cadmium at concentrations of 1 mM provoked m i s p a i r i n g between hypoxanthine and u r a c i l , but not between hypoxathine and adenine nucleotides (14). In a d d i t i o n to i n c r e a s i n g the number of mispairings between uncomplementary n u c l e o t i d e s , c a r c i n o g e n i c metals a l t e r the s t r u c t u r e of the double stranded DNA h e l i x . In the presence of magnesium, c a l c i u m , z i n c , or manganese the secondary s t r u c t u r e of poly A (polyadenosine monophosphate) i n s o l u t i o n c o n s i s t s of both s i n g l e and double h e l i x e s , but i n the presence o f n i c k e l and c o b a l t , poly A appears only as a s i n g l e stranded h e l i x (15). Copper and cadmium r e s u l t i n the formation of randomly c o i l e d " s t r u c t u r e s o f poly A (15). Thus, some potent i a l l y c a r c i n o g e n i c metals appear to a l t e r the secondary s t r u c t u r e of p o l y n u c l e o t i d e s , as well as promoting the m i s p a i r i n g of uncomplementary n u c l e o t i d e s . C o l l e c t i v e l y , these studies suggest that the i n t e r a c t i o n o f p o t e n t i a l l y c a r c i n o g e n i c or mutagenic metal ions with s o l u t i o n s o f p o l y n u c l e o t i d e s causes uncomplementary base p a i r i n g s and d i s t o r t i o n of the secondary o r t e r t i a r y s t r u c ture of DNA. These e f f e c t s i n v i t r o can be c o r r e l a t e d with the i n vivo s i t u a t i o n , since a l t e r a t i o n s o f t h i s nature could r e s u l t in a s i g n i f i c a n t increase i n DNA mutation f r e q u e n c i e s . The m i s p a i r i n g of uncomplementary n u c l e o t i d e s would, f o r the most p a r t , cause point mutations when the DNA i s r e p l i c a t e d . The end r e s u l t of t h i s type of mutation, assuming the mistake was not r e p a i r e d , would be the s u b s t i t u t i o n o f an i n c o r r e c t amino a c i d i n the p r i mary s t r u c t u r e of a c e l l u l a r c o n s t i t u e n t or enzyme. I f a number of unrepaired point mutations o c c u r , i t i s conceivable that a

American Chemical Society Library 1155 16th St. N. w. Martell; Inorganic Chemistry and Medicine Washington, D. inC.Biology 20038 ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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s u f f i c i e n t number o f a l t e r a t i o n s i n c e l l u l a r p r o t e i n s would r e s u l t i n c e l l transformation. This transformed c e l l could then p r o l i f e r a t e abnormally, and with proper promotion, develop i n t o a neoplasm. A d d i t i o n a l experiments have suggested that c a r c i n o g e n i c or mutagenic metals may decrease f i d e l i t y o f DNA r e p l i c a t i o n o r transcription. The f i d e l i t y o f DNA t r a n s c r i p t i o n i s assayed as follows: a s y n t h e t i c template i s mixed with the DNA polymerase enzyme i s o l a t e d from a b a c t e r i a or a v i r u s . Other reagents r e q u i r e d f o r the t r a n s c r i p t i o n o f DNA are added, i n c l u d i n g two r a d i o l a b e l e d triphosphate n u c l e o t i d e s ; one^complementary to the strand being r e p l i c a t e d and l a b e l e d with [ H ] , and the other uncomplementary and l a b e l e d with [ C ] . Using t h i s system i n v e s t i gators have found that the presence o f 1-10 mM b e r y l l i u m (2+) r e s u l t e d i n an increased m i s i n c o r p o r a t i o n o f the uncomplementary c y t o s i n e triphosphate i n t o n u c l e o t i d e s copied from poly A - T (16). These r e s u l t s could have been a t t r i b u t e d i n part to exonuclease contamination o f the DNA polymerase preparation used. The c o n taminating exonuclease would incorporate c y t o s i n e by r e p a i r r a t h e r than by r e p l i c a t i o n . However, Si rover and Loeb (17) found that b e r y l l i u m decreased the f i d e l i t y o f DNA polymerase with a s i m i l a r system using avian myoblast v i r u s DNA polymerase which lacks the exonuclease r e p a i r enzyme. T h e i r r e s u l t s suggest that b e r y l l i u m s a l t s a c t u a l l y reduce the f i d e l i t y o f t r a n s c r i p t i o n . The uncomplementary c y t o s i n e was incorporated once f o r every 1,100 complementary dTTP n u c l e o t i d e s polymerized i n t o the daughter strand using a poly-A o l i g o dT template and natural c o n d i t i o n s . When b e r y l l i u m was added to t h i s mixture the e r r o r frequency increased form 1 per 1,100 to 1 per 75 (17_). In these s t u d i e s b e r y l l i u m was found to bind with the DNA polymerase enzyme; t h u s , t h i s i n t e r a c t i o n probably caused the decrease i n the f i d e l i t y o f transcription. A d d i t i o n a l studies showed that c o b a l t , manganese, and n i c k e l could s u b s t i t u t e f o r magnesium as c o f a c t o r s o f DNA polymerase enzyme (18_). However, even i n the presence o f magnesium, a d d i t i o n s o f c o b a l t and manganese s i g n i f i c a n t l y impaired the f i d e l i t y o f t r a n s c r i p t i o n . Various c a r c i n o g e n i c and mutagenic metals such as Ag, Be, Cd, C r , Cu, Mn, N i , and Pb decreased the f i d e l i t y of DNA t r a n s c r i p t i o n , r e s u l t i n g i n the enhanced i n c o r p o r a t i o n o f the uncomplementary n u c l e o t i d e (19). In c o n t r a s t , s o l u b l e s a l t s o f A l , Ba, Ca, F e , K, Mg, Na, Rb, S r , and Zn d i d not a f f e c t the f i d e l i t y o f DNA synthesis even a t concentrations high enough to i n h i b i t the synthesis o f DNA (19). These biochemical s t u d i e s h i g h l i g h t two important concepts: 1) they i l l u s t r a t e that the mechanism o f metal carcinogenesis f o r a wide v a r i e t y o f c a r c i n o g e n i c o r mutagenic metals may i n v o l v e a decrease i n the f i d e l i t y o f DNA r e p l i c a t i o n ; 2) these s t u d i e s have suggested that t h i s biochemical t r a n s c r i p t i o n system can be used as a screen f o r the d e t e c t i o n o f p o t e n t i a l l y carcinogenic or mutagenic environmental metal carcinogens. The problem with t h i s system as a screen i s the requirement t h a t the metals be i n


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s o l u t i o n to r a p i d l y i n t e r a c t i n v i t r o with the DNA polymerase during the a l l o t t e d assay time of 30-60 minutes. Many carcinogeni c molecules are not r e a d i l y water s o l u b l e . In a d d i t i o n , i f they undergo d i s s o l u t i o n , they may be chemically changed from the o r i g i n a l molecule. For example, when c r y s t a l l i n e n i c k e l subs u l f i d e i s allowed to s o l u b i l i z e i n water, i t forms n i c k e l oxide and n i c k e l hydroxide. It can be more r a p i d l y d i s s o l v e d i n d i l u t e a c i d s o l u t i o n , f o r example HC1, but the adveous product i s N i C l ^ * not the o r i g i n a l n i c k e l compound. Carcinogenic or mutagenic metal s a l t s have varying e f f e c t s on the i n i t i a t i o n of new RNA c h a i n s . S a l t s of Co (2+), Cd (2+), Cu (2+), Mn (2+), and Pb (2+) enhance the rate of i n i t i a t i o n of new RNA chains at concentrations that i n h i b i t e d o v e r a l l RNA synthesis rates (20). However, non-carcinogenic metals such as K , L i , M g , Na^Tand Z n i n h i b i t e d the i n i t i a t i o n of new RNA chains at concentrations which depressed o v e r a l l DNA synthesis (20). Since the metals which have been shown to be c a r c i n o g e n i c or mutagenic are able to a c t i v a t e points of RNA i n i t i a t i o n at concentrations which i n h i b i t t o t a l RNA s y n t h e s i s , while nonc a r c i n o g e n i c metals are not able to a c t i v a t e new RNA chain i n i t i a t i o n at concentrations which are i n h i b i t o r y to RNA synthesis i t i s presumed that t h i s technique may be u t i l i z e d to measure the c a r c i n o g e n i c or mutagenic a c t i v i t y o f s p e c i f i c metal s a l t s (20). These observations also point to other p o s s i b l e mechanisms by which metals cause mutations and/or cancer i n c e l l s . The c a r c i n o g e n i c metals appear to i n i t i a t e new RNA chains and t h e r e fore may lead to the expression o f new genes which under normal c o n d i t i o n s would not be expressed. Moreover, the c a r c i n o g e n i c metals may cause the excessive i n i t i a t i o n and expression of s e l e c t e d gene products. +

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E f f e c t s of Carcinogenic Metals i n B a c t e r i a l

Systems

M i c r o b i a l studies have demonstrated a l i n k between c a r c i n o genesis and mutagenesis and w i l l be reviewed b r i e f l y with reference to the i n d i v i d u a l metals t e s t e d . The metal compound most studied i n b a c t e r i a l mutagenesis systems i s chromium. Hexavalent chromium i s a potent mutagen i n several b a c t e r i a l systems. V e n i t t and Levy (21) t e s t e d the mutag e n i c i t y of three chromate compounds: Na2Cr04, K2Cr04, andCaCr04 using various b a c t e r i a l s t r a i n s . A l l three chromium compounds in concentrations from 0.05 to 0.2 mM per p l a t e y i e l d e d p o s i t i v e mutation frequencies approximately 3 times greater than the c o n t r o l l e v e l (21). the hexavalent chromium ions bind to G-C base p a i r s and r e s u l t i n t r a n s i t i o n s from G-C to A-T when the DNA i s r e p l i c a t e d ( 2 J J . Other studies have also confirmed that hexavalent chromium i s mutagenic i n a v a r i e t y o f b a c t e r i a l systems. In a d d i t i o n to hexavalent chromium i o n s , other metal ions have induced mutations i n b a c t e r i a . These metals include As (3+), Cd (2+), Hg (2+), Mo (6+), Se (4+), Te (4+), Te (6+), and



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V(4+) (22., 23, 21, 25J. These r e s u l t s have a l s o shown that the metal ions l i s t e d above were mutagenic i n a wide v a r i e t y o f bact e r i a l s t r a i n s (22^23,24^25). However, c e r t a i n c a r c i n o g e n i c metal ions were not mutagenic i n b a c t e r i a l systems. The most important o f these carcinogens was the n i c k e l i o n , which d i d not induce mutations i n a v a r i e t y o f the b a c t e r i a l systems t e s t e d (25). The b a c t e r i a l mutagenesis assay i s useful f o r d e t e c t i n g p o t e n t i a l organic carcinogens, but appears to have l e s s value i n assessing the carcinogenic a c t i v i t y of metal carcinogens. A major problem with b a c t e r i a l systems i s that the microsomal a c t i v a t i o n system i s not present and must be added to a c t i v a t e the procarcinogen to the ultimate carcinogen. This introduces considerable v a r i a t i o n s i n the assay. The i n f l u e n c e of m i c r o somal enzyme systems on metal mutagenesis or carcinogenesis has not been well studied and requires f u r t h e r i n v e s t i g a t i o n . Studies i n C e l l

Culture Systems

Introduction. In recent years the use o f t i s s u e c u l t u r e has become one of the most important techniques i n the determination of the t o x i c and c a r c i n o g e n i c a c t i v i t y o f x e n o b i o t i c s . Morphol o g i c a l transformation and c y t o l o g i c a l changes are induced i n t i s s u e c u l t u r e f o l l o w i n g exposure to c a r c i n o g e n i c metals and t h e i r compounds. Tissue c u l t u r e systems have c e r t a i n advantages over m i c r o b i a l assays and i n vivo systems because: 1) the c e l l s are s i m i l a r to those found i n v i v o , but u n l i k e the i n vivo s i t u a t i o n a homogenous population i s t r e a t e d , 2) n e o p l a s t i c transformation i s induced i n t i s s u e c u l t u r e , while m i c r o b i a l systems detect mutations, 3) c o n t r o l o f v a r i a b l e s i n t i s s u e c u l t u r e systems i s much simpler than with i n vivo systems. Tissue c u l t u r e systems are e s p e c i a l l y s u i t e d f o r assaying potent i a l l y carcinogenic metals s i n c e metal carcinogens, u n l i k e the o r g a n i c s , are thought to be primary carcinogens. Metals are thought to be able to i n t e r a c t d i r e c t l y with t a r g e t t i s s u e s , r e q u i r i n g metabolic a c t i v a t i o n f o r cancer i n d u c t i o n , whereas organic carcinogens g e n e r a l l y r e q u i r e a c t i v a t i o n by microsomal enzymes. Exposure of t i s s u e c u l t u r e c e l l s to many organic carcinogens w i l l induce transformation only i f the a c t i v a t i n g enzymes are present. This i s a drawback to the use o f t i s s u e c u l t u r e methods f o r c e r t a i n types of carcinogenesis research with organic chemicals. A c t i v a t i o n by microsomal enzymes, with i n o r g a n i c metal c a r c i n o g e n s , may not play a primary r o l e i n the carcinogenesis process i f c u r r e n t hypotheses are c o r r e c t . However, the p r e l i m i n a r y data presented i n t h i s paper suggests that microsomal enzymes may, i n f a c t , a l t e r the course o f metal carcinogenesis i n t i s s u e c u l t u r e . The e f f e c t s o f metals on t i s s u e c u l t u r e systems w i l l be divided into 3 sections. The f i r s t s e c t i o n i n v o l v e s t o x i c e f f e c t s of the same metals on c e l l c u l t u r e s ; the second s e c t i o n w i l l discuss the e f f e c t s o f metals on t r a n s c r i p t i o n a l , t r a n s l a t i o n a l ,


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and mutagenic processes; and the t h i r d s e c t i o n w i l l d i s c u s s the i n v i t r o transformation o f c e l l s i n c u l t u r e by exposure to metal carcinogens.


Toxic

Effects

Metals have been shown to have a v a r i e t y o f t o x i c e f f e c t s on c e l l s in culture. In one study (26), exposure of r a t embryo muscle c e l l s to n i c k e l s u b s u l f i d e r e s u l t e d i n a depression of c e l l d i v i s i o n and induced abnormal m i t o t i c s p i n d l e s , d i s t o r t e d b i p o l a r s p i n d l e s , lagging chromosomes, and unequal cytoplasmic d i v i s i o n (26). M i t o t i c a r r e s t occurred i n telephase, and the post-telephase period was c o n s i s t e n t with a mechanism of a c t i o n i n v o l v i n g a disturbance i n f u n c t i o n of the m i t o t i c s p i n d l e mechanism (26). Chromosomal breakage was observed i n leukocyte c u l t u r e s a f t e r a 2 day exposure to sodium arsenate (Na^HAsO^ t concentrations from 0.1-10 yg/ml (27_,28). The abberations i n c u r r e d by exposures of leukocytes i n v i t r o to a r s e n i c were s i m i l a r to those observed i n vivo with p a t i e n t s who were t r e a t e d c h r o n i c a l l y with a r s e n i c compounds (27,28.29^). The c l i n i c a l p i c t u r e included a v a r i e t y of abnormal" m i t o t i c and chromosomal c o n f i g u r a t i o n s i n the leukocytes of humans t r e a t e d with a r s e n i c (27,28,29). In a s i m i l a r study A s , Sb, and Te s a l t s , but not Be, C 3 7 C o " , " T e , Hg, N i , Se, and V s a l t s caused chromosomal abberations in human leukocytes (30). Exposure of human leukocytes to cadmium s u l f i d e induced numerous a l t e r a t i o n s w i t h i n the chromosomal s t r u c t u r e (31). Further experimentation showed that the exposure of t i s s u e c u l t u r e f i b r o b l a s t s to CdS04 caused chromosomal breakage and aberrations (32). Other studies have demons t r a t e d that treatment of e u k a r y o t i c c e l l s with A s , Cd, C r , N i , Sb, and Te a l t e r s chromosomal s t r u c t u r e s (13,31,35). These r e s u l t s suggest that the metal compounds ( p a r t i c u l a r l y a r s e n i c , cadmium, chromium, and n i c k e l ) a l t e r e d the normal m i t o t i c processes. An a d d i t i o n a l study has shown that exposure of Chinese Hamster Ovary (CHO) c e l l s to n i c k e l s u b s u l f i d e , a potent c a r c i n o g e n , induced elongation of these c e l l s (36). Following exposure of Chinese Hamster Ovary c e l l s to t h i s n i c k e l carcinogen, the c e l l shape changes from a rounded form to an elongated f i b r o b l a s t i c - ! i k e c e l l s t r u c t u r e (36). The changes i n the morphology of CHO c e l l s resembled those that were caused by exposure o f these c e l l s to agents that elevated c e l l u l a r cAMP levels (36). a

Mutagenic

Effects

A r s e n i c competes with phosphate f o r i n c o r p o r a t i o n i n t o precursors of DNA or RNA such as dATP, rATP, dGTP, rGTP, e t c . (28,37_,38.,|9). If a r s e n i c i s incorporated i n t o molecules such as ATP, then i t may a l s o be added to s e r i n e and threonine residues of c e l l u l a r p r o t e i n s , since p r o t e i n kinase uses the terminal phosphate o f ATP i n the phosphorylation o f a v a r i e t y of c e l l u l a r proteins.



52

INORGANIC

CHEMISTRY IN BIOLOGY A N D M E D I C I N E

Hexavalent chromium was shown to i n h i b i t DNA synthesis i n hamster f i b r o b l a s t s at concentrations o f 0.1 mM (40_,4JL). In these s t u d i e s i t was found that hexavalent chromium was more e f f i c i e n t than t r i v a l e n t chromium i n the i n h i b i t i o n o f DNA s y n t h e s i s , and that the e f f e c t s on t i s s u e c u l t u r e c e l l s o f hexavalent chromium were more pronounced i n a s a l t s / g l u c o s e s o l u t i o n r a t h e r than a complete media, since the l a t t e r had f e t a l bovine serum and other undefined f a c t o r s that promoted the reduction of hexavalent chromium to t r i v a l e n t chromium (40). D i f f e r e n t studies have confirmed that other c a r c i n o g e n i c m e t a l s , such as s o l u b l e s a l t s of n i c k e l , i n h i b i t e d DNA synthesis i n tissue culture c e l l s . These are i n agreement with the s t u d i e s conducted i n vivo demonstrating that c e r t a i n c a r c i n o g e n i c metals a l s o i n h i b i t hepatic DNA synthesis during l i v e r regeneration. Transformation o f C e l l s i n Tissue Culture by Carcinogenic Metals and T h e i r Compounds Mammalian c e l l c u l t u r e s have been used as the basis o f several systems i n d e t e c t i n g the p o t e n t i a l carcinogenic a c t i v i t y of chemicals. B a s i c a l l y , two general approaches have been utilized: continuous c e l l l i n e s and primary c e l l c u l t u r e s . Cell l i n e s have the advantage o f ease o f use, i n that c u l t u r e s do not have to be obtained fresh from animals p r i o r to each t e s t , but may be maintained f o r months to years by proper s u b c u l t u r i n g techniques. They have the disadvantage o f possessing one or more "transformed" c h a r a c t e r i s t i c s ( e . g . , i m m o r t a l i t y ) . In some cases c e l l l i n e s may a l s o l a c k c e r t a i n enzyme systems required f o r metabolic a c t i v a t i o n of chemicals. Some o f the c e l l l i n e s used f o r transformation assays include the murine (BALB/3T3) A31 system (42), and the baby kidney-21 (BHK-21) systems (43). Primary c e l l c u l t u r e s have advantages over c e l l l i n e s i n that the c e l l s are not i n i t i a l l y immortal, and u s u a l l y have none of the transformed c h a r a c t e r i s t i c s which may be seen i n some c e l l l i n e s (41,45). A d d i t i o n a l l y , the embryonic c e l l s which are most widely used f o r transformation t e s t s g e n e r a l l y maintain enzyme systems c h a r a c t e r i s t i c of the o r i g i n a l host f o r at l e a s t a few subcultures. Primary c e l l c u l t u r e s may have the disadvantage, however, of r e q u i r i n g harvesting and preparation of c e l l s p e r i o d i c a l l y , or f o r each t e s t . T h i s problem can be minimized by prepa r a t i o n o f l a r g e numbers of c e l l s and c r y o p r e s e r v a t i o n of known s e n s i t i v e samples. A useful i n v i t r o transformation assay which may be p a r t i c u l a r l y a p p l i c a b l e f o r metals, i s the the enhancement o f hamster embryo c e l l transformation by simian adenovirus. Casto, et al (46) and DiPaolo et al (47) reported that a l l metal s a l t s (of a s e r i e s o f 38 tested) with known c a r c i n o g e n i c a c t i v i t y , increased the frequency of simian adenovirus SA-7 induced transformations.


3.

COSTA E T A L .

Metal

53

Carcinogenesis

Metals were d i v i d e d i n t o three groups: high ( p o s i t i v e at 0.05 mM), moderate ( p o s i t i v e at 0.05 to 0.6 mM), and low ( p o s i t i v e only at concentration greater than 0.9 mM) a c t i v i t y . Activity High

Moderate


Low

Salts

of

Antimony, A r s e n i c , Cadmium, Chromium, Platinum B e r r y ! i u m , C o b a l t , Copper, Lead, Mangagnes, Mercury, N i c k e l , S i l v e r , T h a l l i u m , Zinc Iron

In every c e l l c u l t u r e based transformation assay, several c h a r a c t e r i s t i c s are monitored as i n d i c a t o r s of p o t e n t i a l oncogenic transformation. These c h a r a c t e r i s t i c s , f o r transformed and nontransformed c e l l s , are summarized i n the f o l l o w i n g statements. Normal c e l l s : 1) grow i n an o r d e r l y fashion with l i t t l e c e l l c r i s s - c r o s s i n g , 2) are incapable of forming 3 dimensional c o l o n i e s i n s o f t agar media, and 3) do not form tumors when administered to athymic "nude" mice. The ultimate t e s t of n e o p l a s t i c transformation i s the a b i l i t y of c e l l s to form tumors in "nude" mice. A recent study has shown that the various c r i t e r i a which have been a p p l i e d to t r a n s f o r m a t i o n , such as disordered growth, plant l e c t i n a g g l u t i n a t i o n , and growth i n s o f t agar do not n e c e s s a r i l y i n d i c a t e n e o p l a s t i c t r a n s f o r m a t i o n ; that i s , the a b i l i t y of c e l l s to form tumors when administered to experimental animals (48). A number o f i n v e s t i g a t o r s have t r e a t e d e s t a b l i s h e d c e l l l i n e s with c a r c i n o g e n i c metals and found that morphological transformation was induced i n these c u l t u r e s . Fradkin et al (49) found that treatment of c e l l l i n e s with hexavalent chromium r e s u l t e d i n disordered growth of baby hamster kidney (BHK-12) c e l l s exposed to 0.25 or 0.5 yg/1 of CaCrO^ The c h a r a c t e r i s t i c change i n the growth pattern of these c e l l s was a l o s s of order in c o l o n i e s and extensive c e l l p i l i n g which was not present i n untreated c u l t u r e s (49). A f t e r the c e l l s were t r e a t e d with CaCHty, they acquired the a b i l i t y to grow i n a s e m i s o l i d media. Normal c e l l s were not able to p r o l i f e r a t e i n t h i s media. Treatment of c u l t u r e s of mouse f e t a l c e l l s with CaCr0 r e s u l t e d in morphological a l t e r a t i o n s (35). C e l l s were exposed to CrCl3 or K C r 0 , and exposures to both hexavalent and t r i v a l e n t chromium compounds r e s u l t e d i n c o l o n i e s of c e l l s that p i l e d up i n a randomly o r i e n t e d fashion surrounded by various giant c e l l s (35). The authors of t h i s study questioned whether or not the morphologi c a l a l t e r a t i o n s induced by these chromium compounds represented any n e o p l a s t i c changes (35). 4

2

2

7



54

INORGANIC

CHEMISTRY IN BIOLOGY A N D

MEDICINE

In a s e r i e s of studies by Costa et al (12,50^57), secondary c u l t u r e s of Syrian hamster f e t a l c e l l s were exposed to e i t h e r c r y s t a l l i n e n i c k e l s u b s u l f i d e or amorphous n i c k e l s u l f i d e . Following exposure to the n i c k e l compounds the c u l t u r e s were washed free of the metals and the c e l l s seeded to form c o l o n i e s . Treatment with the potent c a r c i n o g e n , n i c k e l s u b s u l f i d e , induced a concentration dependent incidence of morphological t r a n s f o r m a t i o n , while s i m i l a r treatment with amorphous n i c k e l s u l f i d e , the non-carcinogen, d i d not r e s u l t i n any of these changes. The n i c k e l s u b s u l f i d e transformed c o l o n i e s were c l o n e d , derived i n t o immortal c e l l l i n e s , and tested f o r t h e i r a b i l i t y to grow i n s o f t agar and to form tumors i n athymic "nude" mice. A l l morphologically transformed c e l l l i n e s tested were able to p r o l i f e r a t e i n s o f t agar and formed tumors i n "nude" mice. S i m i l a r c l o n i n g of normal c u l t u r e s was u n s u c c e s s f u l , and none of the normal mass c u l t u r e s derived from hamster embryos, and t e s t e d f o r colony formation i n s o f t agar or formation of tumors in "nude" mice were p o s i t i v e . Materials

and Methods

Test Compounds. Nickel s u b s u l f i d e ( c r y s t a l l i n e a N i 3 S , p a r t i c l e s i z e < 5 ym) was provided by Dr. Edward K o s t i n e r , U n i v e r s i t y of C o n n e c t i c u t , and i t s p u r i t y and c r y s t a l s t r u c t u r e were v e r i f i e d by emission spectroscopy and X-ray d i f f r a c t o m e t r y as p r e v i o u s l y described (2^58j. Amorphous n i c k e l monosulfide (NiS) was p r e c i p i t a t e d by a d d i t i o n of ammonium s u l f i d e to a s o l u t i o n of Ni CI 2 that was prepared from c a r b o n y l - d e r i v e d Ni dust and u l t r a p u r e HC1. The amorphous NiS was devoid of c r y s t a l s t r u c t u r e , based upon X-ray d i f f r a c t o m e t r y . The a N i S and NiS powders were s t e r i l i z e d by washing i n acetone immediately p r i o r to suspension i n t i s s u e c u l t u r e medium. Morphological Transformation Assay Using Syrian Hamster Fetal c e l l s . Syrian hamster embryo c e l l s were i s o l a t e d as p r e v i o u s l y described (50_,51_,52_). T e r t i a r y passage c u l t u r e s were prepared by p l a t i n g about 1 X 10 c e l l s i n t o 100 mm diameter p l a t e s . The c e l l s were allowed to attach to the monol a y e r f o r one or two days and s e l e c t e d c u l t u r e s were then pretreated with 3 yg/ml o f benzopyrene f o r 24 h. Pretreatment of c e l l s with 2 yg/ml of benzopyrene was shown to increase the microsomal p r o t e i n content by 2 f o l d w i t h i n 24 h i n these c e l l s (data not shown). Cultures were then t r e a t e d with the appropriate metal compounds three times f o r a period of two days for each treatment. Following t h i s procedure c e l l s were removed from the monolayer by t r y p s i n i z a t i o n (0.25% t r y p s i n i n Puck's s a l i n e A ) , and 5,000 and 10,000 c e l l s were r e p l a t e d to form c o l o n i e s i n 100 mm diameter p l a t e s c o n t a i n i n g 10 ml of Dulbecco's medium supplemented with 10% f e t a l bovine serum (Hy-clone, S t e r i l e Systems, I n c . ) . Foci assays were conducted by p l a t i n g a p p r o x i mately 50,000 t r e a t e d or untreated c e l l s i n t o 100 mm diameter tissue culture plates. The media was replenished about one time 2

3

2

6


3.

COSTA E T A L .

Metal

55

Carcinogenesis

each week during the subsequent two week incubation p e r i o d . Cultures seeded to form c o l o n i e s were incubated 12-14 days. C e l l s that were seeded f o r the f o c i assay were incubated f o r 23 days. At the end o f the appropriate incubation period the monolayer of c e l l s was washed two times with a phosphate buffered normal s a l i n e s o l u t i o n , f i x e d with 95% e t h a n o l , and stained with a 0.5% (w/v) c r y s t a l v i o l e t - e t h a n o l mixture. Where a p p r o p r i a t e , the t o t a l number of s u r v i v i n g c o l o n i e s were counted i n each p l a t e . Each colony or f o c i was evaluated f o r morphological transformation using a l i g h t microscope by an observer who was not aware of the treatment c o n d i t i o n s . Mutagenesis Assay Using Chinese Hamster Ovary C e l l s . Chinese Hamster Ovary c e l l s were grown i n monolayer c u l t u r e w i t h i n a humidified atmosphere composed of 5% C 0 and 95% a i r using McCoy's 5a medium supplemented with 10% f e t a l bovine serum (Hy-clone, S t e r i l e Systems, I n c . ) . Cultures of Chinese Hamster Ovary c e l l s were exposed to the appropriate metal compounds f o r 24 h and then allowed to undergo approximately twelve d i v i s i o n s (doubling time 14-16 h) p r i o r to being placed i n the s e l e c t i o n ^ media. At the end o f t h i s d i v i s i o n period approximately 7 X 10 c e l l s (monolayer c u l t u r e s of each had formed) were incubated i n the s e l e c t i o n medium c o n s i s t i n g of complete McCoy's media supplemented with 3 yg/ml 8-azaguanine (8-AG) and 6 yg/ml 6-thioguanine (6-TG). Cultures were incubated i n the s e l e c t i o n media f o r approximately 3 weeks. The s e l e c t i o n medium was replaced with fresh media c o n t a i n i n g the 8-AG and 6-TG every three o r four days during the s e l e c t i o n p e r i o d . At the end of t h i s i n c u b a t i o n , c e l l s were f i x e d with 95% ethanol and stained with a 0.5% (w/v) c r y s t a l v i o l e t - e t h a n o l mixture. The number of mutant c o l o n i e s i n each p l a t e was then counted. By d e f i n i t i o n a colony c o n s i s t e d of 50 or more c e l l s i n a c l u s t e r . As expected, nearly a l l of the c e l l s i n the p l a t e s died o f f , l e a v i n g only a few c o l o n i e s i n p l a t e s t r e a t e d with mutagenic agents. These procedures are s i m i l a r to other mutation assays reported i n the l i t e r a t u r e (59). T o x i c i t y t e s t s were conducted i n Chinese Hamster Ovary c e l l s to determine the c o n c e n t r a t i o n o f e i t h e r Ni*3S o r NiS which a f f e c ted c e l l p l a t i n g e f f i c i e n c y f o l l o w i n g a 24 h exposure p e r i o d . P r o l i f e r a t i n g c u l t u r e s of Chinese Hamster Ovary c e l l s were exposed to the n i c k e l compounds f o r 24 h , and then the c e l l s were t r y p s i n i z e d from the monolayer. C e l l numbers were d e t e r mined with a hemocytometer and 400 c e l l s were p l a t e d to form c o l o n i e s i n 100 mm diameter t i s s u e c u l t u r e p l a t e s . The c e l l s were incubated f o r about 9 days, f i x e d , s t a i n e d , and the t o t a l number of s u r v i v i n g c o l o n i e s i n each p l a t e were counted and expressed as a f u n c t i o n o f the t o t a l number of c e l l s p l a t e d .


2

2

Uptake

Studies

Log phase c u l t u r e s were prepared i n Leighton Tubes (tubes c o n t a i n i n g a p l a s t i c microscopic s l i d e which provides a surface f o r c e l l growth). C e l l s were exposed to the metal compunds


56

INORGANIC CHEMISTRY

IN

BIOLOGY A N D

MEDICINE


and f o l l o w i n g the exposure period were washed two times with normal s a l i n e , f i x e d with 95% ethanol and s t a i n e d with a c r y s t a l v i o l e t s o l u t i o n (0.5% c r y s t a l v i o l e t i n e t h a n o l ) . The c e l l s were observed with a l i g h t microscope f o r the presence of i n t r a c e l l u l a r n i c k e l compounds. For e l e c t r o n microscopy s t u d i e s , c e l l s were prefixed i n 3% gluteraldehyde s o l u t i o n buffered with 0.05 M Phosphate Buffer pH 7.4 c o n t a i n i n g 0.05 M sucrose. C e l l s were r i n s e d i n 0.05 M Phosphate Buffer pH 7.4 c o n t a i n i n g 0.5 M sucrose f i v e times f o r 10 min and then dehydrated using sequential ethanol and acetone washes. The c e l l s were embedded i n an epoxy r e s i n mixture and then sectioned with a microtone. The s e c t i o n s were post stained with Uranyl Acetate and Lead C i t r a t e and then examined with an e l e c t r o n microscope. Results Morphological Transformation o f Syrian Hamster Fetal C e l l s . Figure 1 shows the o r d e r l y growth pattern of c e l l s i n a normal untreated colony of Syrian hamster f e t a l c e l l s . In c o n t r a s t , Figure 2 shows the changes i n the growth pattern of normal c e l l s r e s u l t i n g from treatment with a metal carcinogen such as c r y s t a l l i n e Ni*3S2. Note that the transformed c e l l s grew i n a d i s o r d e r l y p a t t e r n , with c e l l s invading each o t h e r ' s boundaries. This n e t - l i k e , disordered growth pattern i s the morphological a l t e r a t i o n c h a r a c t e r i s t i c of n e o p l a s t i c transformation. Our l a b o r a t o r y has cloned a number of c o l o n i e s having disordered growth patterns s i m i l a r to those shown i n Figure 2 (50,51,52). These clones were shown to produce tumors i n athymic "nude"" mice f o l lowing subcutaneous i n j e c t i o n and to form 3 dimensional c o l o n i e s i n soft agar medium (50,51,52). C e l l s having a normal growth pattern as shown i n Figure 1 were administered to "nude" mice and p l a t e d to form c o l o n i e s i n s o f t agar medium. However, these c e l l s d i d not produce tumors i n "nude" mice, or 3 dimensional c o l o n i e s i n s o f t agar medium. Concentration Dependent Morphological Transformation b y N i ^ Table 1 shows the r e s u l t s of a t y p i c a l experiment where c u l t u r e s of Syrian hamster embryo c e l l s were exposed to several concentrat i o n s o f NioS or NiS. Control c u l t u r e s , which were untreated or t r e a t e d with amorphous NiS, had no s i g n i f i c a n t incidence of morphological t r a n s f o r m a t i o n . However, the incidence of Ni3S2 induced morphological transformation was dependent upon the concentration of Ni*3S which the c e l l s were exposed. 2

2

Influence of Benzopyrene Pretreatment on Metal Induced Transformation. The i n d u c t i o n o f cancer by metal carcinogens i s thought to be independent o f microsomal enzyme activation. These conclusions are not based upon experimental evidence as such, but on an idea that has developed from at l e a s t two o b s e r v a t i o n s : 1) carcinogenic metals and t h e i r com-


Metal

Carcinogenesis


COSTA E T A L .

Figure 1.

Photograph showing the ordered growth pattern of a typical "normal" colony


INORGANIC



58

Figure 2.

Photograph showing the disordered growth pattern of a Ni S formed colony 3

2


trans-

3.

COSTA E T A L .

Metal

Carcinogenesis

59

TABLE I EFFECT OF CRYATALLINE N i S AND AMORPHOUS NiS ON THE TRANSFORMATION OF SYRIAN HAMSTER FETAL CELLS IN TISSUE CULTURE


3

Test Compound

Controls aNi S 0

NiS

0

2

Concentration ug/ml 0

No. o f , Plates 3

of

Proportion Transformed Colonies^

36

2/2045 [0.1%]

O.l 1.0 5.0 10.0

12 12 12 6

25/599^ 40/584 26/27l 4/20

[4.2%] [6.8%] [8.9%] [20%]

0.1 1.0 5.0

6 6 6

1/203 0/261 2/291

[0.5%] [0.0%] [1.0%]

c

c

5 , 0 0 0 c e l l s were plated i n t o 35 mm t i s s u e c u l t u r e p l a t e s colonies. a

to form

Ratio o f the number o f transformed c o l o n i e s to the number o f t o t a l c o l o n i e s on a l l c u l t u r e p l a t e s (with percentage i n brackets []) c

P < 0.005 versus corresponding r a t i o f o r control p l a t e s


2 [x t e s t ] .

60

INORGANIC


pounds are r e l a t i v e l y simple molecules which are not e x t e n s i v e l y metabolized i n v i v o , and 2) the i n d u c t i o n o f cancer by a carcinogenic metal occurs at the exposure s i t e even i n t i s s u e s , such as muscle l a c k i n g the c a p a b i l i t y to a c t i v a t e and metabolize drugs, as well as other f o r e i g n compounds. In Table 2 we present some p r e l i m i n a r y data suggesting that induction o f microsomal enzymes, i n p a r t i c u l a r cytochrome P 448 associated hydroxylases, enhanced the N i S induced transformat i o n o f Syrian hamster embryo c e l l s . Cultures t r e a t e d w i t h N i S or benzopyrene had incidences o f transformation i n the colony assay ranging from 3.2-3.6% o f the s u r v i v i n g c o l o n i e s when 10,000 t r e a t e d c e l l s were challenged to form c o l o n i e s . However, c u l t u r e s pretreated with benzopyrene and then exposed to N i S had considerably higher proportion of morphologically transformed colonies. In t h i s experiment, c u l t u r e s were pretreated with benzopyrene using a s i n g l e 24 h exposure i n t e r v a l while the same c u l t u r e s were exposed to the metal compounds f o r 2 days using 3 separate exposures. The enhancement of N i S transformation i n c u l t u r e s pretreated with benzopyrene was greater than the incidence of transformation induced by each agent alone. These r e s u l t s suggested that benzopyrene induces a process which enhanced the carcinogenesis o f N i S . It i s p o s s i b l e that t h i s i n d u c i b l e process represents the a c t i v a t i o n o f a r y l hydrocarbon hydroxylases, and t h e i r subsequent i n t e r a c t i o n with the N i S enhances i t s c a r c i n o g e n e s i s . 3

2

3


3

3

3

2

2

2

2

3

2

T o x i c i t y of NJ3S2 and Benzopyrene i n S y r i a n Hamster Embryo Cells. Table 3 shows the e f f e c t on the p l a t i n g e f f i c i e n c y o f Syrian hamster embryo c e l l s exposure to NiS or N i 3 S 2 . Note that exposure o f c e l l s to 2 pg/ml o f Ni'3S2 reduced c e l l p l a t i n g e f f i c i e n c y by o h e - h a l f the values obtained i n untreated c u l t u r e s . Results from other experiments which we have conducted suggests that a s i n g l e 24 h treatment of c e l l s with 3 yg/ml of benzopyrene only reduces c e l l p l a t i n g e f f i c i e n c y by approximately 30%. It appears from the data shown that pretreatment with benzopyrene enhanced the N i ' S induced t o x i c i t y to Syrian hamster c e l l s greater than the t o x i c i t y d i s p l a y e d f o r each compound i n d i v i d u a l ly. However, the N i S c a r c i n o g e n i c enhancement by benzopyrene was g r e a t e r than the t o x i c enhancement. 3

2

3

2

Mutagenesis of Chinese Hamster Ovary C e l l s by N i S . Prel i m i n a r y experiments (Table 4) suggest that Ni*3S2, a potent carcinogen has weak to no mutagenic a c t i v i t y i n Chinese Hamster Ovary c e l l s . The incidence of c o l o n i e s r e s i s t a n t to the t o x i c i t y of 6-thioguanine and 8-azoguanine was not s i g n i f i c a n t l y increased i n c u l t u r e s that had been pretreated with Ni^Sg f o r 24 h. When Chinese Hamster Ovary c e l l s were t r e a t e d with NiS (a nonc a r c i n o g e n ) , a l e s s e r degree of r e s i s t a n c e to the s e l e c t i n g agents was found. Chinese Hamster Ovary c e l l s were exposed f o r 24 h to various l e v e l s of the NiS or N i S to determine the 3

3

2

2



2

Control

(3 yg/ml)

(untreated)

Benzopyrene

NiS (2 yg/ml)

3

0/171 == 0%

1/72 = 1.4%

(N=2)

(N=2)

(N=3)

28/305 = 9.2%

Ni S

(2 yg/ml)

(N=3)

7/34 = 21%

2

N i S (2 yg/ml)and Benzopyrene (3 yg/ml pretreatment f o r 24 h)

3

Plated

5,000 C e l l s

Treatment Conditions

0/336 = 0%

20/559 = 3.6%

1/308 = 0.3%

(N=2)

(N=4)

(N=2)

(N=4)

(N=4)

20/92 = 22%

16.494 = 3.2%

Plated

Colonies

10,000 C e l l s

Proportion of Transformed

TABLE II EFFECT OF PRETREATMENT WITH BENZOPYRENE UPON THE TRANSFORMATION OF SYRIAN HAMSTER EMBRYO CELLS BY NICKEL COMPOUNDS


(N=4)

(N=3) (N=3) (N=4) (N=2)

5.3

1.0

2.0 0

0

No. o f Transformed Foci per P l a t e


62

INORGANIC

CHEMISTRY IN

BIOLOGY A N D

MEDICINE

TABLE III EFFECT OF BENZOPYRENE PRETREATMENT ON PLATING EFFICIENCY IN CULTURES SUBSEQUENTLY TREATED WITH Ni~ S Treatment Conditions

Plating Efficiency

5,000

Cells

(%)

10,000

Cells

Control (no treatment)

1.60%

1.71%

Ni S

0.89%

0.71%

NiS (2 yg/ml)

1.54%

0.72%

NioS2 (2 yg/ml)

0.23%

0.21%

3

2

(2 yg/ml)

and benzopyrene (3 yg/ml pretreatment f o r 24 h)

T h i r d passage log phase c u l t u r e s of Syrian hamster f e t a l c e l l s were t r e a t e d as described i n the t a b l e . Cultures treated with benzopyrene and Ni3S2 were exposed to benzopyrene f o r 24 h p r i o r to treatment with the metal. Cultures were t r e a t e d with the metal compounds three times using a two-day exposure f o r each treatment. C e l l s were then removed from the p l a t e by t r y p s i n i z a t i o n and the number of c e l l s present i n each p l a t e was d e t e r mined with a hemocytometer. Five thousand or ten thousand c e l l s were r e p l a t e d to form c o l o n i e s i n t o 100 mm diameter plates and the number o f s u r v i v i n g c o l o n i e s i n each p l a t e was counted. Each number shown i n the t a b l e i s the mean of four t i s s u e c u l t u r e plates.


3.

Metal

COSTA E T A L .

63

Carcinogenesis

TABLE IV


MUTAGENESIS OF CHINESE HAMSTER OVARY CELLS BY SPECIFIC NICKEL COMPOUNDS 24h treatment Condition

Concentration (yg/ml)

No treatment

Crystalline

0.2+0.4

(N=5)

0.5 1.0 1.5

0.5 + 0.58 0.5 + 0.58 1.3 + 1.53

(N=4) (N=4) (N=4)

0.5 1.0 1.5

1.0 + 0.8 2.3 + 0.5 2.5 + 1.0

(N=4) (N=4) (N=4)

0

Amorphous NiS

Ni S 3

2

Number o f 6-TG and 8-AG Resistant Colonies per Plate x + sd

P r o l i f e r a t i n g c u l t u r e s of Chinese Hamster Ovary c e l l s were exposed f o r 24 h to the compounds shown i n the t a b l e . The c e l l s were allowed to undergo twelve d i v i s i o n s i n complete growth medium and then about 7 X 10^ c e l l s attached to the monolayer were placed i n s e l e c t i o n medium (McCoy's 5a medium supplemented with 10% f e t a l bovine serum, 8 yg/ml 8-azaguanine (8-AG) and 6 yg/ml 6-thioguanine (6-TG). Cultures were incubated i n s e l e c t i o n medium f o r about three (3) weeks. The s e l e c t i o n medium was replenished with fresh medium about two (2) times each week. At the end of the s e l e c t i o n period the c u l t u r e s were washed two (2) times with normal s a l i n e , f i x e d with 95% ethanol and stained with an e t h a n o l - c r y s t a l v i o l e t s o l u t i o n (0.5% w/v c r y s t a l v i o l e t i n 95% e t h a n o l ) . A colony was defined as a c l u s t e r of 50 or more cells.


64

INORGANIC

CHEMISTRY IN BIOLOGY A N D M E D I C I N E

t o x i c i t y o f these compounds. These r e s u l t s are shown i n Table 5 and show that concentrations of NioS 2 ranging from the L D t o LDgQ were used i n attempting to obtain mutations. 5 0

Uptake of N i S


3

2

Into C e l l s

Figures 3 and 4 show l i g h t microscope photographs o f CHO and Syrian hamster embryo c e l l s r e s p e c t i v e l y which have phagoc y t i z e d Ni3S2 p a r t i c l e s . Table 6 shows that Syrian hamster embryo c e l l s a c t i v e l y take up Ni0S2 p a r t i c l e s and undergo morphological transformation f o l l o w i n g exposure to t h i s compound. S i m i l a r exposure to amorphous NiS r e s u l t s i n no s i g n i f i c a n t transformation and l i t t l e uptake o f Ni S p a r t i c l e s . Table 7 shows that the approximate h a l f l i f e of Ni3S2 p a r t i c l e s i n c e l l s i s 40 h. The p a r t i c l e s may be a l t e r e d to a form not v i s i b l e with the l i g h t microscope or r e s u l t i n c e l l l y s i s . Discussion The development o f an i n v i t r o metal carcinogenesis t e s t system which i s r e l i a b l e , r a p i d , and inexpensive has been the subj e c t of several recent reports (47^48,50-57). These r e s u l t s were discussed i n e a r l i e r s e c t i o n s o F t J T i s chapter. The purposes of the present report i s to review some of the work conducted on the e f f e c t s o f metals using i n v i t r o a s s a y s , and to r e l a t e these f i n d i n g s s p e c i f i c a l l y to c u r r e n t views on the mechanisms of metal c a r c i n o g e n e s i s . The p r e l i m i n a r y data presented i n t h i s chapter suggests some new points of view r e l a t i n g to the c a r c i n o g e n e s i s of metals using i n v i t r o systems. The experiments suggest that the benzopyrene pretreatment enhances the t o x i c i t y and c a r c i n o g e n i c i t y o f Ni*3S . The enhancement of transformation by combined treatment of c e l l s with Ni*3S and benzopyrene i s greater than the summation o f that induced by i n d i v i d u a l compounds. Further work i s required to c l a r i f y the mechanisms involved i n the enhancement. These a d d i t i o n a l studies may i n v o l v e a study o f the e f f e c t o f microsomal enzymes on metal carcinogenesis. The p r e l i m i n a r y benzopyrene experiments described i n t h i s paper have important i m p l i c a t i o n s i n assessing c a r c i n o g e n i c hazards a s s o c i a t e d with human exposure to metal carcinogens. If smokers (benzopyrene i s found i n c i g a r e t t e smoke as well as other inducers of microsomal enzymes) are exposed to metal carcinogens, the r e l a t i v e r i s k s of c o n t r a c t i n g neoplasms of the r e s p i r a t o r y systems are g r e a t e r i n these i n d i v i d u a l s than i n those who do not smoke. 2

2

The a d d i t i o n a l p r e l i m i n a r y data concerned the p o s s i b l e mutagenic a c t i v i t y o f N i S , Current t h e o r i e s concerning p o s s i b l e modes o f cancer i n d u c t i o n favor a c e l l u l a r mutation or a s e r i e s of mutagenic events f o r the i n i t i a t i o n o f n e o p l a s t i c t r a n s f o r mations. The data reported here i n d i c a t e s that Ni'3S2 d i s p l a y s l i t t l e mutagenic a c t i v i t y . However, f u r t h e r experiments are 3

2


Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980. 1 17 22 34 36 60 30 41 44 59

5 68 88 136 144 240 120 164 176 236

30.0 10.0 3.0 1.0 0.3 0.1 30.0 10.0 3.0 0.3

93

373

Plate

P l a t i n g E f f i c i e n c y (%) No. S u r v i v i n g Colonies per 400 c e l l s p l a t e d

0

per

Colonies

No. o f S u r v i v i n g

AND AMORPHOUS NiS IN CHINESE HAMSTER OVARY CELLS

P r o l i f e r a t i n g monolayer c u l t u r e s of Chinese Hamster Ovary c e l l s were exposed f o r 24 h to the c o n c e n t r a t i o n of the compounds shown i n the t a b l e . The monolayer of c e l l s were dislodged by treatment with t r y p s i n , counted, and then 400 c e l l s were r e p l a t e d to form c o l o n i e s . The c u l t u r e s were allowed to form c o l o n i e s f o r about 2 weeks with media changes two times each week. The p l a t e s were then f i x e d , s t a i n e d , and the number of s u r v i v i n g c o l o n i e s per p l a t e were counted.

Amorphous NiS

Ni,S,

(no treatment)

Crystalline

Control

24 h Treatment Conditions

0

Exposure Concentration (yg/ml)

0

TOXICITY OF CRYSTALLINE N i S

TABLE V



INORGANIC


Figure 3. Light microscope photograph of Chinese hamster ovary cells that have phagocytized Ni S particles. Note the vacuoles and dark Ni S particles in some of the vacuoles. 3

Figure 4.

2

3

2

Light microscope photograph of Syrian hamster embryo cells that have phagocytized Ni S particles 3

2


3.

COSTA E T A L .

Metal

Carcinogenesis

67

TABLE VI PHAGOCYTOSIS AND MORPHOLOGICAL TRANSFORMATION BY N i S AND NiS IN SYRIAN HAMSTER EMBRYO CELLS 3

2


Chemicals Amorphous NiS

Crystalline Ni S 3

Concentration (yg/ml)

Transformed Colonies Total S u r v i ing Colonies

Phagocytosis (Percentage of c e l l s having nickel particles

1

0/222 (0%)

0.10%

5

0/189 (0%)

0.40

10

0/166 (0%)

0.80

1

6/214 (2.8%)

6.70

5

12/138 (8.7%)

23.60

10

11/93 (11.8%)

42.90

2

Secondary c u l t u r e s o f S y r i a n hamster embryo c e l l s were exposed to the p a r t i c u l a t e n i c k e l compounds shown i n the t a b l e f o r three separate exposure f o r a period of 48h (transformation assay) o r 24h (uptake a s s a y ) . To assess morphological t r a n s f o r mation the free metal compounds were removed from contact with normal s a l i n e . The c e l l s were then dislodged from the monolayer by t r y p s i n i z a t i o n , and r e p l a t e d (1,000-5,000 c e l l s ) i n t o 100 mm t i s s u e c u l t u r e p l a t e s to form c o l o n i e s . Following 12 days o f incubation the c o l o n i e s were f i x e d , s t a i n e d , and evaluated f o r morphological transformation. The number of transformed c o l o nies was expressed as a f u n c t i o n of the t o t a l number of s u r viving colonies. Each transformation r a t i o represents the mean of 6 separate p l a t e s . For the uptake s t u d i e s , log-phase monol a y e r c u l t u r e s grown on p l a s t i c microscopic s l i d e s were exposed to the metal compounds. Following the exposure period the c e l l s were washed two times with normal s a l i n e , f i x e d with 95% ethanol, and stained with a m e t h a n o l - c r y s t a l v i o l e t s o l u t i o n . One thousand c e l l s were examined with a l i g h t microscope (see Figure 2 and 3) i n each s l i d e f o r the presence o f n i c k e l p a r t i c l e s .



68

INORGANIC

CHEMISTRY IN BIOLOGY A N D

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TABLE VII HALF-LIFE OF M 3 S 2 PARTICLES IN SYRIAN HAMSTER EMBRYO CELLS C e l l s with nickel

intracellular particles

Time a f t e r removal of f r e e metal

67.3

0 h

66.4

4 h

50.8

8 h

43.8

24 h

27.6

48 h

6.7

96 h

3.0

120 h

Log phase Hamster embryo c e l l s were exposed to 20 yg/ml o f N i ' S for 24 h. Following t h i s exposure the media c o n t a i n i n g N i 3 S was removed and the c e l l s were washed two time with normal s a l i n e . The c e l l s were then placed i n f r e s h complete media and at various time i n t e r v a l s were f i x e d , s t a i n e d and 1,000 c e l l s examined with the l i g h t microscope f o r i n t r a c e l l u l a r nickel p a r t i c l e s . Each number shown i n the t a b l e i s the mean o f 2 s l i d e s where a t o t a l o f 2,000 c e l l s were examined f o r both s l i d e s . The c e l l s having n i c k e l p a r t i c l e s are expressed as a percentage o f those examined. 3

2

2



3.

Metal

COSTA E T A L .

69

Carcinogenesis

required t o evaluate the mutagenic a c t i v i t y o f Ni3S2» one o f the most potent metal carcinogens. Other s t u d i e s (40,41) have suggested that several c a r c i n o g e n i c metals are mutagenic i n b a c t e r i a l systems, but n i c k e l compounds have shown no mutagenic a c t i v i t y i n b a c t e r i a l systems. Very few studies have evaluated the mutagenic a c t i v i t y o f carcinogenic metals i n mammalian c e l l c u l t u r e systems. Most o f these studies have demonstrated e f f e c t s on chromosomal s t r u c t u r e and f u n c t i o n (26-35). I t i s important to note that i n a l l o f our s t u d i e s we have used amorphous NiS (a non-carcinogen) as a negative c o n t r o l i n a d d i t i o n t o an untreated c o n t r o l . Amorphous NiS was n e i t h e r c a r c i n o g e n i c nor mutagenic i n our two t e s t systems. In using the Syrian hamster embryo system f o r metal c a r c i n o genesis t e s t i n g i t was important to demonstrate that n e o p l a s t i c changes were associated with morphological transformation (52). A d d i t i o n a l l y , when t e s t i n g various metal samples f o r c a r c i n o g e n i c a c t i v i t y , i t i s important t o use p o s i t i v e (NT3S2) and negative (NiS) c o n t r o l s i n every experiment t o evaluate the c o n s i s t e n c y of each assay. I f Ni So does not induce morphological t r a n s f o r mation i n a concentration dependent manner, then the v a l i d i t y o f the e n t i r e assay should be suspect. S i m i l a r l y , i f untreated c u l tures and NiS t r e a t e d c u l t u r e s have a high incidence o f morphol o g i c a l transformation the r e s u l t s o f the assay are not v a l i d . 3

A p o s s i b l e explanation f o r why N i 3 $ 2 i s a potent carcinogen while amorphous NiS lacks a c t i v i t y was presented i n the uptake studies. From other experiments i t appears that the c a r c i n o g e n i c a c t i v i t y o f p a r t i c u l a t e metal compounds i s p r o p o r t i o n a l t o t h e i r c e l l u l a r uptake. In future s t u d i e s we hope t o concentrate on t h i s phagocytosis as a p o s s i b l e mechanism o f metal induced carcinogenesis.

Abstract. We have reviewed work conducted in our laboratory and other laboratories investigating the carcinogenic and mutagenic effects of metals and their compounds upon in vitro systems. Preliminary data is also presented which shows the following: 1) Pretreatment of Syrian hamster embryo c e l l s with benzopyrene, an inducer of aryl hydrocarbon hydroxylase, potentiates the morphological transformation of Syrian hamster embryo cells induced by Ni S . The incidence of N i S transformation in cultures pretreated with benzopyrene was in some instances 10 fold greater than those transformations caused by similar exposure to either N i S or benzopyrene alone. 2) Ni S treatment of Chinese Hamster Ovary c e l l s caused the appearance of 2-3 6-thioguanine and 8azoguanine resistant colonies (per plate, 7 X 10 c e l l s plated) while untreated Chinese Hamster Ovary c e l l s averaged 0.2 resistant colonies per plate for a similar number of c e l l s at risk. Therefore, N i S displays very weak or no mutagenic a c t i v i t y in the mutagenesis system tested, and 3) carcinogenic a c t i v i t y of 3

2

3

3

2

2

3

2

6

3

2


70

INORGANIC


particulate metal compounds such as N i S is proportional to their cellular uptake. Cells actively phagocytized particulate Ni S but did not take up amorphous NiS particles to a significant degree. The latter observation may help understand why specific metal compounds are carcinogenic. 3

2

3

2

ACKNOWLEDGMENT This work was supported by grant #ES02254 from the National I n s t i t u t e of Environmental Health S c i e n c e s .


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