Environmental Epidemiology - American Chemical Society

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11 Research Strategy for Assessing Human Health Risks from Exposure to DNA-Reactive Chemicals 1,3-Butadiene as a Case Study James A . Bond, Leslie R e c i o , and Roger O . M c C l e l l a n Chemical Industry Institute of Toxicology, P.O. Box 12137, Research Triangle Park, NC 27709

The interaction of chemicals or their metabolites with DNA is a major factor in chemical carcinogenesis. One potential strategy for more accurately estimating human health risks from

exposure to

DNA

-reactive chemicals is to develop research that will improve our understanding of the mechanisms of action of chemicals within an exposure --> tissue dose --> cancer response paradigm. The development of quantitative linkages between exposure and response, which are based on biologically plausible mechanisms of action at exposure levels likely to be encountered by people, will significantly improve the risk assessments for human exposures to DNA-reactive

chemicals.

1,3-Butadiene (BD) represents an interesting case study in which the foregoing research strategy is providing data that are critical for understanding interspecies differences in responses to BD. BD is a carcinogen in rats and mice; mice are more sensitive than rats. It is not known whether BD poses a carcinogenic risk for humans. BD requires metabolic activation to DNA-reactive

epoxides that can bind

to DNA to initiate a series of events leading to tumor formation.

Spe-

cies differences in activation and detoxication need to be considered in developing human risk estimates for BD. BD

activation-to-detox-

ication ratios are markedly different for mouse (74), rat (6), and human (6) liver tissues. The differences in the ratios between mice and rats are consistent with the higher carcinogenic sensitivity of mice to BD, compared with rats. Additional data, which were developed by using a transgenic mouse model system, have indicated that BD induces in vivo mutations in target tissues. These data coupled with the BD metabolism data in mice, rats, and humans form the basis of a mechanistically based BD tissue dosimetry model in rats and mice

0065-2393/94/0241-0137$08.00/0 © 1994 American Chemical Society

Draper; Environmental Epidemiology Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

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ENVIRONMENTAL EPIDEMIOLOGY that can now serve as the basis for a human dosimetry model. This dosimetry model enables key extrapolations from high- to low-dose exposures and between species and will be employed in a human risk assessment for

BD.


T H E INTERACTION OF CHEMICALS OR THEIR METABOLITES WITH DNA is a major factor i n carcinogenesis of a n u m b e r of chemicals ( J , 2, a n d references therein). A c e n t r a l hypothesis for the m e c h a n i s m of action of m a n y D N A - r e a c t i v e c h e m i c a l carcinogens is that the generation of D N A damage c o u p l e d w i t h subsequent c e l l r e p l i c a t i o n can result i n mutations i n specific genes that play a k e y role i n the carcinogenic process. H o w e v e r , not a l l types of damage to D N A , w h e t h e r i n the form of D N A adducts, cross-links, or base deletions, w i l l have the same impact o n the r e p l i c a t i n g c e l l . F o r exa m p l e , some abundant a n d stable D N A adducts are excellent markers of exposure b u t may be poor markers of risk, because they are not p r o m u t a genic. D N A adducts may occur at sites that are not essential for c e l l function and thus w o u l d have little impact o n cancer d e v e l o p m e n t . A d d i t i o n a l l y , the ability of a c e l l to repair the damaged D N A a n d the rate a n d fidelity of the repair w i l l have i m p o r t a n t consequences for w h e t h e r the damage is fixed and u l t i m a t e l y results i n a m u t a t i o n a l event. T h u s , k n o w l e d g e of h o w c h e m icals interact w i t h D N A , the p r o b a b i l i t y of that interaction i n d u c i n g a specific m u t a t i o n , a n d the subsequent p r o b a b i l i t y of t u m o r formation is critical for the d e v e l o p m e n t of biologically based models to p r e d i c t the h u m a n h e a l t h consequences for D N A - r e a c t i v e chemicals. A major goal of the C h e m i c a l I n d u s t r y Institute of Toxicology ( C U T ) D N A - r e a c t i v e c h e m i c a l research strategy is the d e v e l o p m e n t of a b i o l o g i cally based mechanistic approach to assessing h u m a n cancer risks for exposure to these types of chemicals. T h e research involves the d e v e l o p m e n t of biologically based descriptions of d o s i m e t r y (i.e., m e t a b o l i s m a n d h e m o g l o b i n - a n d D N A - a d d u c t formation) a n d i n c o r p o r a t i o n of mechanisms of c h e m ically i n d u c e d m u t a t i o n into a biologically based framework for t u m o r formation. Several components to the D N A - r e a c t i v e c h e m i c a l research strategy are used at C U T . T h e s e components, i l l u s t r a t e d i n F i g u r e 1, i n c l u d e b i o markers, dosimetry, D N A interaction, m u t a t i o n i n d u c t i o n , c e l l proliferation, and t u m o r i n d u c t i o n . D a t a d e v e l o p e d f r o m b i o m a r k e r , dosimetry, a n d D N A interaction studies serve as critical i n p u t for the d o s i m e t r y models that are u l t i m a t e l y d e v e l o p e d . D a t a d e v e l o p e d from D N A interaction studies as w e l l as m u t a t i o n i n d u c t i o n , c e l l proliferation, a n d t u m o r i n d u c t i o n studies, c o u p l e d w i t h the d o s i m e t r y models, can support the d e v e l o p m e n t of biological response models. A r e c o m m e n d e d research approach for s t u d y i n g D N A - r e a c t i v e c h e m i cals includes experiments that quantitatively describe the translocation of material from the external e n v i r o n m e n t to critical target sites on D N A a n d



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Figure 1. Important components of a DNA-reactive chemical research strategy designed with a biologically based mechanistic approach for assessing human cancer risks for exposure to these types of chemicals.

the role of the damaged D N A i n increasing the l i k e l i h o o d of m u t a t i o n . T h e s e experiments i n v o l v e s u c h d y n a m i c processes as a b s o r p t i o n , d i s t r i b u t i o n , a n d i n the case of nonreactive chemicals, m e t a b o l i s m to D N A - r e a c t i v e a n d stable nontoxic c o m p o u n d s . T h e f o r m u l a t i o n of quantitative descriptions of the metabolic pathways i n v o l v e d i n the p r o d u c t i o n of D N A - r e a c t i v e metabolites s h o u l d be a k e y c o m p o n e n t of the approach. T h e s e descriptions i n c l u d e b o t h activation a n d detoxication pathways, because the u l t i m a t e concentration of the D N A - r e a c t i v e species at the target site depends o n the balance b e t w e e n these pathways. It is l i k e l y that b o t h exposure c o n c e n t r a t i o n a n d exposure rate changes can affect the balance b e t w e e n activation a n d detoxication p r o cesses. T h e rates of D N A damage (i.e., D N A adduct formation a n d D N A strand breaks) a n d r e p a i r as w e l l as the t i m e course for formation a n d p e r sistence of adducts are also major considerations. A d d i t i o n a l l y , k n o w l e d g e of c e l l r e p l i c a t i o n can p r o v i d e a critical linkage b e t w e e n i n t e r a c t i o n of c h e m i cals w i t h D N A a n d the ability of the d a m a g e d D N A to i n d u c e m u t a t i o n a l events a n d subsequent t u m o r d e v e l o p m e n t . A n evaluation of the use of m a c r o m o l e c u l a r adducts f o r m e d f r o m reactive chemicals as monitors of exposure, i n w h i c h the use of the b i o m a r k e r s as a n i n d i c a t o r of tissue dose is p a r t i c u l a r l y e m p h a s i z e d , is another i m p o r t a n t consideration. T h e m a c r o m o l e c u l a r adducts s h o u l d i n c l u d e D N A adducts a n d h e m o g l o b i n adducts. U l t i m a t e l y , this research strategy w i l l specify the relationships a m o n g different b i o m a r k e r s of exposure w i t h respect to t h e i r relative levels a n d t i m e of appearance after exposure to D N A - r e a c t i v e chemicals.

BD as a Case Study B D represents an i n t e r e s t i n g case study i n w h i c h the foregoing research strategy is p r o v i d i n g data that are c r i t i c a l for u n d e r s t a n d i n g interspecies dif-



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ferences i n responses to B D . B D , a m o n o m e r w i d e l y u s e d i n the p r o d u c t i o n of synthetic r u b b e r a n d other resins, exhibits l o w acute i n h a l a t i o n toxicity i n rodents (hC^ < 120,000 p p m ) (3) a n d causes sensory i r r i t a t i o n i n h u m a n s at concentrations of 2000-8000 p p m (4). O c c u p a t i o n a l exposures to B D c a n result d u r i n g p r o d u c t i o n , storage, a n d transport of the c h e m i c a l . B D has b e e n detected i n cigarette smoke (5) a n d a u t o m o b i l e exhaust (6) a n d is c u r r e n t l y l i s t e d as one of the 189 hazardous air pollutants i n the 1990 C l e a n A i r A c t A m e n d m e n t s (7). B D induces tumors at m u l t i p l e organ sites i n B 6 C 3 F 1 m i c e a n d S p r a g u e - D a w l e y rats (8-10). I n m i c e , several tissues are targets for B D carcinogenicity, i n c l u d i n g heart, l u n g , m a m m a r y g l a n d , ovary, forestomach, b o n e marrow, a n d liver. A n activated K-ras gene was o b s e r v e d i n l u n g a n d l i v e r tumors a n d l y m p h o m a s of B 6 C 3 F 1 m i c e exposed to B D (11). T h e striki n g aspect of B D - i n d u c e d carcinogenicity is the h i g h sensitivity of m i c e to B D . T u m o r s w e r e o b s e r v e d i n m i c e at concentrations as l o w as 6 p p m , a n d steep c o n c e n t r a t i o n - r e s p o n s e curves w e r e e v i d e n t for several t u m o r s . T h e t u m o r sites i n rats are different a n d i n c l u d e the t h y r o i d , m a m m a r y g l a n d , Z y m b a l g l a n d (auditory subaceous gland), u t e r u s , testis, a n d pancreas. Rats exhibit a r e l a t i v e l y l o w sensitivity to B D , because tumors occur at B D c o n centrations (1000-8000 p p m ) nearly three orders of m a g n i t u d e h i g h e r t h a n in mice. B D displays mutagenic activity i n Salmonella typhimurium, b u t o n l y i n the presence of hepatic 9000-g supernatant (12-14), w h i c h indicates that B D is not mutagenic b u t that its metabolites, possibly l , 2 - e p o x y b u t - 3 - e n e [ b u tadiene monoepoxide ( B M O ) ] a n d l , 2 : 3 , 4 - d i e p o x y b u t a n e [butadiene d i e poxide ( B D E ) ] , are responsible for the o b s e r v e d bacterial mutagenicity. B D is also genotoxic i n v i v o , i n d u c i n g c h r o m o s o m e aberrations a n d sister c h r o m a t i d exchanges i n b o n e m a r r o w cells a n d m i c r o n u c l e u s f o r m a t i o n i n p e r i p h e r a l b l o o d of male B 6 C 3 F 1 m i c e (15,16) b u t not i n S p r a g u e - D a w l e y rats (16). Studies o n the i n d u c t i o n of B D - i n d u c e d m u t a t i o n a l events have p r i m a r i l y b e e n c a r r i e d out i n c e l l c u l t u r e systems, a n d extrapolation of these data to i n v i v o genetic effects is difficult. T h e r e f o r e , i n v i v o approaches to d e t e r m i n e m u t a t i o n i n d u c t i o n i n somatic cells of tissues n e e d to be c o n s i d e r e d . Transgenic m i c e constructed w i t h m u t a t i o n a l target genes can p e r m i t the study of d o s e - r e s p o n s e a n d the m o l e c u l a r basis of m u t a t i o n i n tissues f r o m animals w i t h d e f i n e d carcinogenic exposures. C e r t a i n transgenic mouse models are i n v i v o shuttle vector systems (17) that use bacterial genes (e.g., lad or lacZ) as m u t a t i o n a l targets i n s e r t e d w i t h i n the X-phage g e n o m e that is integrated i n t o the mouse g e n o m e . T h e i n d u c t i o n of mutations can b e d e t e r m i n e d w i t h respect to exposure, doseto-target tissues, a n d D N A adduct levels, t h e r e b y p r o v i d i n g a l i n k b e t w e e n i n v i v o exposures a n d mutagenic events. T h e m u t a t e d transgene can be seq u e n c e d , t h e r e b y p r o v i d i n g the o p p o r t u n i t y for assessment of m u t a t i o n a l


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specificity. T h i s provides a u n i q u e o p p o r t u n i t y to d e t e r m i n e the i n d u c t i o n of B D mutations i n m i c e that w i l l reflect the integrated i n v i v o p h a r m a c o k i n e t i c a n d biotransformation processes that u l t i m a t e l y result i n B D - d e r i v e d mutagenic metabolites. T h r e e e n z y m e s appear to play major roles i n the o v e r a l l m e t a b o l i s m of B D , c y t o c h r o m e P - 4 5 0 - d e p e n d e n t monooxygenases, epoxide hydrolases, and glutathione ( G S H ) S-transferases. I n a d d i t i o n to e n z y m i c reactions, B D epoxides m a y n o n e n z y m a t i c a l l y h y d r o l y z e o r conjugate w i t h G S H . P r e s ently, insufficient data quantitatively describe the c o n t r i b u t i o n of these v a r ious pathways i n S p r a g u e - D a w l e y rats a n d B 6 C 3 F 1 m i c e , species susceptib l e to B D - i n d u c e d carcinogenesis, or h u m a n s , a species p o t e n t i a l l y at risk for B D . Because one or m o r e of the B D epoxides may p l a y a role i n the carcinogenicity of B D , a quantitative d e t e r m i n a t i o n of the balance of a c t i vation (i.e., epoxide formation) a n d i n a c t i v a t i o n (i.e., epoxide h y d r o l y s i s or conjugation) is essential for i m p r o v i n g o u r u n d e r s t a n d i n g a n d assessment of h u m a n risk f o l l o w i n g exposure to B D . T h i s chapter describes quantitative species differences i n the oxidation of B D a n d B M O b y c y t o c h r o m e P 4 5 0 - d e p e n d e n t monooxygenases a n d i n the inactivation of B M O b y epoxide hydrolases a n d glutathione S-transferases f o u n d b y u s i n g m i c r o s o m a l a n d cy tosolic preparations of livers o b t a i n e d f r o m S p r a g u e - D a w l e y rats, B 6 C 3 F 1 m i c e , transgenic m i c e ( C D 2 F 1 ) , a n d h u m a n s . I n a d d i t i o n , transgenic m i c e w e r e u s e d to assess the capacity of B D to i n d u c e i n v i v o mutations. T h e data f r o m these studies p r o v i d e a q u a n t i tative d e s c r i p t i o n of the relative c o n t r i b u t i o n s of the various pathways of B D m e t a b o l i s m i n three a n i m a l species, i n c l u d i n g h u m a n s , a n d the role that m e t a b o l i s m plays i n f o r m i n g mutagenic D N A - r e a c t i v e B D metabolites. D a t a d e v e l o p e d o n B D m e t a b o l i s m can serve as the basis for the d e v e l o p m e n t of a B D d o s i m e t r y m o d e l . T h e B D d o s i m e t r y m o d e l w i l l enable k e y extrapolations from h i g h - to low-dose exposures a n d b e t w e e n species a n d w i l l be p r e d i c t i v e of target tissue levels of reactive B D metabolites. T h e d o s i m e t r y m o d e l can b y e m p l o y e d i n a h u m a n risk assessment for B D .

Materials and Methods Animals. M a l e S p r a g u e - D a w l e y ( C D ) rats (9-10 weeks) a n d male B 6 C 3 F 1 m i c e (9-10 weeks) w e r e o b t a i n e d f r o m C h a r l e s R i v e r Laboratories (Raleigh, N C ) . T h e M u t a M o u s e ( M M ) transgenic strain [ B A L B / c X D B A / 2 (CD2F1)] of m i c e (6 weeks) was p u r c h a s e d from H a z l e t o n R e s e a r c h P r o d ucts, Inc. T h e construction of the shuttle vector i n X-phage (Xgt 10 lacZ), w i t h the i n s e r t e d bacterial target gene for mutagenesis (lacZ) a n d p r o d u c t i o n of the transgenic m i c e , is d e s c r i b e d i n d e t a i l elsewhere (18). A l l animals w e r e d e t e r m i n e d to be free f r o m v i r a l infection a n d w e r e a c c l i m a t e d for at least 2 weeks p r i o r to use. A n i m a l s w e r e fed w i t h standard d i e t ( N I H - 0 7 ; Z e i g l e r B r o t h e r s , G a r d n e r s , PA) a n d r e c e i v e d w a t e r a d l i b i t u m . T h e y w e r e


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m a i n t a i n e d on a 12-h l i g h t - d a r k cycle, b e g i n n i n g at 0700, a n d h o u s e d at 22 ± 2 °C a n d 55 ± 5% relative h u m i d i t y .

Preparation of Liver Microsomes for Metabolism Studies.

Ro-

dents w e r e e u t h a n i z e d b y e i t h e r s o d i u m p e n t o b a r b i t a l or C 0 asphyxiation, a n d the livers w e r e excised, frozen i n l i q u i d n i t r o g e n , a n d stored at — 80 °C. L i v e r s w e r e slowly t h a w e d w h i l e o n ice, w e i g h e d , cut into pieces, a n d h o m o g e n i z e d w i t h 4 volumes of isotonic K C l - T r i s buffer w i t h six passes of a Teflon-glass h o m o g e n i z e r (1100 revolutions p e r m i n u t e B r a u n ) . M i c r o s o m e s a n d cytosol w e r e p r e p a r e d as d e s c r i b e d p r e v i o u s l y (19).


2

Samples of l i v e r (n = 12) f r o m t r a u m a v i c t i m s (Tennessee D o n o r S e r vices, N a s h v i l l e , T N ) w e r e u s e d i n these e x p e r i m e n t s . Samples w e r e frozen (— 80 °C) a n d r e m a i n e d frozen d u r i n g s h i p m e n t . Tissues w e r e slowly t h a w e d on ice a n d h o m o g e n i z e d , a n d microsomes a n d cytosol w e r e isolated as d e s c r i b e d p r e v i o u s l y (19). Typically, a l l 12 samples w e r e u s e d for the i n i t i a l assessment of reaction rates for an e n z y m e - c a t a l y z e d reaction. T h e 12 s a m ples w e r e t h e n r a n k - o r d e r e d b y e n z y m e activity, a n d the three samples r e p resenting the highest, lowest, a n d m e d i a n e n z y m e activities w e r e u s e d for the e n z y m e k i n e t i c experiments. F o r d e t e r m i n a t i o n of B D oxidation k i n e t ics, a l l 12 l i v e r samples w e r e used. P r o t e i n content was d e t e r m i n e d b y u s i n g a m o d i f i e d m i c r o - L o w r y m e t h o d (20). C y t o c h r o m e P450 content was estimated spectrally (21).

Cytochrome P450-Dependent Metabolism of BD.

Microsomes

w e r e d i l u t e d w i t h 0 . 1 - M phosphate buffer ( p H 7.4) to the d e s i r e d p r o t e i n concentration. O n e m i l l i l i t e r of d i l u t e d microsomes was p l a c e d i n each 10m L v i a l ( H y p o V i a l , P i e r c e C h e m i c a l C o m p a n y , R o c k f o r d , I L ) , w h i c h was sealed w i t h T u f - B o n d septa (Pierce C h e m i c a l C o m p a n y , R o c k f o r d , I L ) . Vials w e r e shaken at 37 °C i n a D u b n o f f metabolic shaking incubator. A f t e r a 10m i n p r e e q u i l i b r a t i o n p e r i o d , B D (600-25,000 p p m ) i n a gas-tight syringe ( H a m i l t o n C o m p a n y , R e n o , N V ) was injected i n t o the sealed, airtight vials. After an a d d i t i o n a l 1 0 - m i n p r e i n c u b a t i o n , the e n z y m i c reaction was started b y a d d i n g 100 |xL r e d u c e d n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate ( N A D P H ) (4 fjimol). P r e l i m i n a r y experiments i n d i c a t e d that this p r e i n c u bation p e r i o d was essential for e q u i l i b r a t i o n of B D b e t w e e n the l i q u i d a n d gas phases. A i r samples (100 u X ) w e r e w i t h d r a w n f r o m the headspace (i.e., gas phase) of the v i a l at 5 - m i n intervals (up to 45 min) a n d a n a l y z e d b y gas chromatography w i t h a H e w l e t t - P a c k a r d gas c h r o m a t o g r a p h ( H P 4890A S e ries II) e q u i p p e d w i t h a flame i o n i z a t i o n detector a n d a 7-ft X 1/8-in stainless-steel c o l u m n f i l l e d w i t h d i p h e n y l p h e n y l e n e oxide (Tenax) 3 5 - 6 0 m e s h . T h e carrier gas was h e l i u m (18.5 m L / m i n ) a n d the detector, c o l u m n , a n d injector temperatures w e r e 250, 130, a n d 200 °C, respectively. U n d e r these conditions, the r e t e n t i o n times of B D a n d B M O w e r e 0.80 m i n a n d 2.20


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m i n , respectively. C a l i b r a t i o n curves of B D a n d B M O w e r e l i n e a r i n the concentration ranges of 10-55,000 p p m a n d 1-340 p p m , respectively.

Enzyme-Mediated Hydrolysis of B M O . E n z y m e - m e d i a t e d h y d r o lysis of B M O b y epoxide hydrolase was m e a s u r e d exactly as d e s c r i b e d for the c y t o c h r o m e P 4 5 0 - d e p e n d e n t reactions, except that B M O was u s e d as the substrate for the reaction a n d N A D P H was not a d d e d . N A D P H was o m i t t e d from the reactions to p r e v e n t e n z y m i c oxidation of B M O . To assess the n o n e n z y m i c hydrolysis of B M O , reactions w e r e c a r r i e d out i n the p r e s ence of heat-inactivated tissue. Enzyme-Mediated Conjugation of B M O with G S H . T h e reaction mixtures (0.1 m L ) c o n t a i n i n g different B M O concentrations (2.5, 7.4, 17.4, 3 7 . 1 , a n d 74.5 m M ) , cytosolic p r o t e i n (1.0 m g / m L ) , a n d G S H (10 m M ) w e r e p l a c e d i n S c r e w C a p S e p t u m vials (Pierce C h e m i c a l C o . , R o c k f o r d , I L ) a n d sealed w i t h T u f - B o n d septa. H - G S H (40 u , C i / m L ) was a d d e d to the vials, a n d after a 1 0 - m i n i n c u b a t i o n at 37 °C, 0.1 m L c o o l e d m e t h a n o l was a d d e d , a n d the p r e c i p i t a t e d proteins w e r e centrifuged for 1 m i n at 12,500 g. R e a c tions w e r e l i n e a r w i t h t i m e for at least 10 m i n . A n aliquot of supernatant (10 |ubL) was chromatographed b y h i g h - p r e s s u r e l i q u i d chromatography ( H P L C ) (Waters 510 p u m p s , M i l f o r d , M A ) . M e t a b o l i t e s w e r e separated o n a 5-|xm C U l t r a s h e r e c o l u m n (250 X 4.6 (i.d.) m m ; B e c k m a n ) a n d the absorbance of the effluent was m o n i t o r e d b y U V detection (Waters 486 T u n a b l e A b s o r b ance Detector, X = 263 nm). M e t a b o l i t e s w e r e q u a n t i t a t e d b y m o n i t o r i n g the radioactivity of the eluant w i t h a P a c k a r d F l o w O n e B e t a i n - l i n e r a d i o f l o w detector (Packard, D o w n e r s G r o v e , I L ) . A n a l y s i s was p e r f o r m e d u n d e r isocratic conditions of methanohO. 1% trifluoroacetic a c i d (15:85) w i t h a 1.0 m L / m i n f l o w rate. T h e r e t e n t i o n times for G S H , o x i d i z e d glutathione ( G S S G ) , a n d the two major G S H conjugates, S-(l-hydroxy-3-buten-2-yl)glutathione a n d S - ( 2 - h y d r o x y - 3 - b u t e n - l - y l ) g l u t a t h i o n e , w e r e 3.8, 4.5, 7.8, a n d 8.5 m i n , respectively. D a t a o n rates of formation of G S H conjugates that are r e p o r t e d represent the s u m of the i n d i v i d u a l rates of formation for each of the conjugates. R e c o v e r y of G S H , G S S G , a n d metabolites f r o m the c o l u m n was greater than 9 9 % . 3

1 8

Experimental Design for Mutagenicity Studies. T h e m u t a g e n i c ity e x p e r i m e n t u t i l i z e d three groups of animals w i t h five M M p e r group. T h e B D group was exposed to 625 p p m B D for 5 consecutive days for 6 h / day. T h i s exposure concentration was selected because previous studies i n dicated that exposure of B 6 C 3 F 1 m i c e to 625 p p m B D for u p to 60 weeks r e s u l t e d i n significant elevation of t u m o r incidences i n a n u m b e r of organs (8). T h e two r e m a i n i n g groups of animals consisted of air controls that w e r e h o u s e d i n chambers similar to those of the B D - e x p o s e d group a n d a group of animals a d m i n i s t e r e d N - e t h y l - N - n i t r o s o u r e a (ENU) at 250 m g / k g (intra-


144


p e r i t o n e a l injection dissolved i n d i m e t h y l sulfoxide) as a positive c o n t r o l . A n i m a l s w e r e h o u s e d i n i n d i v i d u a l w i r e m e s h cages i n s i d e a 1-m H i n n e r s style c h a m b e r (22). D u r i n g the 6 h of exposure to air or B D , the animals w e r e w i t h o u t food b u t h a d free access to water. A i r f l o w i n the c h a m b e r was m a i n t a i n e d w i t h i n 1 0 % of 15 air changes p e r hour.


3

B D exposure levels i n the c h a m b e r w e r e m o n i t o r e d b y infrared spectroscopy u s i n g a F o x b o r o W i l k e s M i r a n I A I R ( N o r w a l k , C T ) . B D was g e n erated d i r e c t l y from a l i q u i d - g a s storage tank operated at approximately 24 p o u n d s p e r square i n c h . T h e v a p o r was passed t h r o u g h a c a l i b r a t e d f l o w c o n t r o l d e v i c e p r i o r to m i x i n g w i t h the high-efficiency particulate a i r - f i l t e r e d d i l u t i o n air s u p p l y i n g the exposure chamber. T h e B D exposure c o n c e n t r a tions throughout the c h a m b e r w e r e w i t h i n 5 % of the target. R e l a t i v e h u m i d i t y i n the c h a m b e r was m a i n t a i n e d at 50 ± 1 0 % , a n d t e m p e r a t u r e was m a i n t a i n e d at 22 ± 2 °C. T h e five c o n t r o l m i c e w e r e s i m i l a r l y h o u s e d i n a second c h a m b e r i n the same r o o m . T h e y w e r e exposed to clean air of the same t e m p e r a t u r e , relative h u m i d i t y , a n d air flow as that d e l i v e r e d to the B D - e x p o s e d animals.

Animal Necropsy and Tissue Collection. M M w e r e k i l l e d b y a n overdose of s o d i u m p e n t o b a r b i t a l a n d exsanguinated b y cardiac p u n c t u r e , a n d the b l o o d was collected i n h e p a r i n i z e d syringes. T h e l i v e r a n d lungs excised from the animals w e r e i m m e d i a t e l y frozen i n l i q u i d n i t r o g e n a n d subsequently stored frozen at — 80 °C. B o n e m a r r o w was r e m o v e d f r o m the tibias a n d femurs b y r i n s i n g w i t h D u l b e c c o s phosphate buffered saline ( G I B C O , G r a n d I s l a n d , N Y ) . T h e c e l l suspension was p l a c e d i n a m i c r o c e n trifuge t u b e , a n d the b o n e m a r r o w cells w e r e p e l l e t e d i n a microcentrifuge at m a x i m u m speed for 3 0 - ^ 5 sec. T h e supernatant was d i s c a r d e d ; the c e l l p e l l e t was frozen i n l i q u i d n i t r o g e n a n d t h e n stored at — 80 °C u n t i l the D N A was extracted. Bacterial Strains and Media. T h e f o l l o w i n g bacterial strains (mcrA~ a n d mcrbB~) w e r e u s e d : Escherichia coli C/lacZ~ lad- (tetracyc l i n e , a m p i c i l l i n ) a n d E. coli C~fh¥JlacL~ (Stratagene, L a Jolla, C A ) . S t a n d a r d bacterial L u r i a - B e r t a n i ( L B ) m e d i u m (21) was u s e d for bacterial g r o w t h a n d p l a t i n g . F o r visualization of plaques, 5 - b r o m o - 4 - c h l o r o - 3 - i n d o l y l - P - D galactoside (X-gal) (0.45 m g / m L ) was i n c l u d e d i n the m e d i u m . D N A Extraction. F r o z e n tissues w e r e t h a w e d i n a p p r o x i m a t e l y 5 m L of lysis buffer ( 1 0 - m M t r i s - H C l p H 8.0, 150 m M N a C l , 2 0 - m M e t h y l enediaminetetraacetic a c i d p H 8.0) a n d carefully h o m o g e n i z e d u s i n g a h a n d h e l d D o u n c e homogenizer. D N A was extracted f r o m tissues b y u s i n g stand a r d techniques (24). T h e concentration of D N A was d e t e r m i n e d b y U V absorption.


11.

BONDETAL.


145

Chemicals


Packaging of X-Phage D N A from the Mouse Genome and Phage Titering. T h e X-phage g e n o m e was " p a c k a g e d " f r o m the mouse g e n o m e b y u s i n g G i g a - P a c k G o l d packaging extract (Stratagene, L a Jolla, C A ) . A p p r o x i m a t e l y 7.5 jjig of mouse genomic D N A (1.5 m g / m L ) was u s e d i n each packaging reaction. Packaging reactions w e r e i n i t i a t e d a c c o r d i n g to the m a n ufacturer's directions, u s i n g a 3-h i n c u b a t i o n at 37 °C. T h e reaction was t e r m i n a t e d b y d i l u t i o n w i t h 1.0 m L of S M buffer ( 5 0 - m M T r i s - H C l , p H 7.5, 1 0 - m M M g S Q , 1 0 0 - m M N a C l , 0 . 0 1 % gelatin). 4

T h e packaging reaction (2.5 u,L) was adsorbed onto a l o g g r o w t h c u l t u r e of E. coli C/lacZ~ b y gently m i x i n g a n d i n c u b a t i n g at r o o m t e m p e r a t u r e for 20 m i n . T h e m i x t u r e was a d d e d to L B m e d i u m w i t h agarose (plus X-gal) a n d p o u r e d onto a 150-mm plate. T h e plates w e r e i n c u b a t e d at 37 °C o v e r n i g h t , a n d the n u m b e r of plaques p e r plate w e r e c o u n t e d . T h e a m o u n t of packaged D N A r e q u i r e d to p r o d u c e approximately 1500 plaques was calculated, as was the n u m b e r of p l a q u e - f o r m i n g units (pfu) p e r m i c r o g r a m of D N A u s e d i n the packaging reaction. A l l packaging reactions y i e l d e d m o r e t h a n 10,000 pfu/jxg D N A .

Determination of lacZ" Mutant Frequency. To d e t e r m i n e the lacZ~ m u t a n t frequency i n b o n e marrow, l u n g , a n d l i v e r tissue samples, D N A was i n d e p e n d e n t l y extracted from each tissue for each a n i m a l . T h e D N A samples for each a n i m a l i n the same exposure g r o u p (five p e r each exposure group) w e r e packaged i n d e p e n d e n t l y into X-phage. A f t e r the d e t e r m i n a t i o n of the pfu for each i n d i v i d u a l D N A sample, an e q u a l n u m b e r of pfu from each i n d i v i d u a l a n i m a l w i t h i n an exposure group w e r e c o m b i n e d to d e t e r m i n e the lacZ~ mutant frequency. T h e r e f o r e , a single lacZ~ m u t a n t frequency was d e t e r m i n e d for each tissue for each exposure group. A total of 1 X 10 X-phage plaques w e r e e x a m i n e d f r o m the b o n e m a r r o w samples, and i n l u n g a n d l i v e r samples, approximately 5 X 10 X-phage plaques w e r e examined. 5

5

A l o g g r o w t h c u l t u r e of E. coli CllacZ' (5 m L ) at 2.5 X 10 b a c t e r i a / m L and a t i t e r e d v o l u m e of packaging reaction ( D N A f r o m five animals) that w o u l d p r o d u c e 4500 plaques w e r e c o m b i n e d i n a 5 0 - m L c u l t u r e t u b e a n d i n c u b a t e d at r o o m t e m p e r a t u r e for 20 m i n . L B m e d i u m w i t h agarose (plus X-gal) was a d d e d to each t u b e , w h i c h was t h e n p i p e t t e d onto three 1 5 0 - m m plates. T h e plates w e r e i n c u b a t e d at 37 °C overnight. 8

T h e total n u m b e r of b l u e plaques (lacZ ) was estimated b y c o u n t i n g a d e f i n e d , o n e - t e n t h area of three plates a n d extrapolating the m e a n to the full plate a n d the n u m b e r of plates for the e n t i r e p l a t i n g . T h e n u m b e r of lacZ~ mutant clear or light b l u e plaques w e r e c o u n t e d a n d " p i c k e d " for confirmation b y r e p l a t i n g o n L B m e d i u m w i t h agarose plus X - g a l . T h e lacZ~ mutant frequency was calculated b y d i v i d i n g the total n u m b e r of c o n f i r m e d mutant plaques b y the total phage p o p u l a t i o n a n a l y z e d a n d was expressed +



146

as lacZ~ m u t a n t s / 1 0 plaques. T h e n u m b e r of o b s e r v e d m u t a n t plaques w i t h i n each tissue group was u s e d to d e t e r m i n e statistical significance r e l a tive to the tissues f r o m the air c o n t r o l g r o u p , a s s u m i n g a Poisson d i s t r i b u tion. 5


Results Mutant Frequency in Different Tissues of Mice following E x posure to B D . T h e lacZ" m u t a n t frequency i n three tissues of m i c e exposed to 619 ± 3.2 p p m B D (mean ± standard d e v i a t i o n [SD]) a n d E N U was d e t e r m i n e d 14 days f o l l o w i n g the last exposure. T h e n u m b e r o f lacZ~ mutant plaques o b s e r v e d , a n d the lacZ~ m u t a n t frequency for each tissue is s h o w n i n Table I. T h e b a c k g r o u n d lacZ~ m u t a n t frequency i n the tissues e x a m i n e d from c o n t r o l m i c e r a n g e d from 2 to 4 m u t a n t s / 1 0 plaques. T h e positive c o n t r o l , E N U , was m u t a g e n i c i n a l l tissues e x a m i n e d . T h e lacZ~ mutant frequency i n the B D - e x p o s e d m i c e d i d not show a significant i n crease i n the b o n e m a r r o w or liver. H o w e v e r , t h e r e was a significant increase i n m u t a n t frequency i n the lungs of exposed m i c e (p < .001). 5

Microsomal Metabolism of B D . T h e i n i t i a l rate of B D oxidation i n l i v e r microsomes was linear w i t h p r o t e i n content over the range of 1 . 0 - 9 . 0

Table I.

lacZ~ Mutant Frequency i n Bone M a r r o w , L u n g , and L i v e r Samples from B D Exposed Animals and Controls 0

No. of Mutants

Tissue Bone marrow Air control BD N-Ethyl-iV-nitrosourea ( E N U ) exposed Lung Air control BD ENU-exposed Liver Air control BD ENU-exposed

Mutant

Frequency (xl0~ ) 5

3 2 71

3.0 1.8 73.l

29 59 88

4.4 9.l W.2

12 18 71

2.4 3.1 13.0

h

b

h

b

BD-exposed mice were exposed to 625 ppm B D (6 h/day for 5 consecutive days). Controls were exposed to air only for the same duration as the BD-exposed mice. Mice dosed with E N U received a single intraperitoneal injection of 250 mg ENU/kg. All mice were killed 14 days after the last inhalation exposure or injection for mutant frequency analysis. ''Significantly greater (p < .001) than air control for each tissue by Poisson analysis of the number of mutant plaques relative to air controls. a


11.

BOND ET AL.


147

Chemicals


m g p r o t e i n / m L a n d w i t h t i m e for u p to 30 m i n (data not shown). F o r the e n z y m e - m e d i a t e d reactions u s i n g l i v e r m i c r o s o m e s , b o t h the disappearance of B D f r o m the gas phase a n d the appearance of B M O i n the gas phase w e r e m e a s u r e d . T h e p r e v i o u s l y d e t e r m i n e d parameters o f B M O m e t a b o l i s m (see further on) w e r e i n c o r p o r a t e d into equations (19) u s e d to describe the i n v i t r o system. T h e equations w e r e t h e n u s e d to estimate the M i c h a e l i s - M e n ten constants for B D oxidation as d e s c r i b e d b y C s a n a d y et a l . (19). T h e m o d e l was f o u n d to adequately describe b o t h B D disappearance f r o m the headspace a n d B M O appearance i n the headspace o f reaction vials c o n t a i n i n g l i v e r microsomes f r o m a l l species. T h e M i c h a e l i s - M e n t e n parameters are l i s t e d i n T a b l e I I . T h e m a x i m u m velocity ( V ) for B D oxidation to B M O for h u m a n l i v e r microsomes was about o n e - h a l f that o b s e r v e d w i t h B 6 C 3 F 1 mouse l i v e r m i crosomes a n d twofold h i g h e r than i n rat l i v e r microsomes (Table II). T h e V for the reaction was about 1.5-fold h i g h e r i n B 6 C 3 F 1 mouse l i v e r m i crosomes, c o m p a r e d w i t h M M l i v e r microsomes. T h e apparent M i c h a e l i s constants ( K s ) for B D oxidation i n l i v e r microsomes from h u m a n s , S p r a g u e - D a w l e y rats, B 6 C 3 F 1 m i c e , a n d M M w e r e s i m i l a r a n d r a n g e d f r o m 2 to 5 u , M . T h e r e w e r e some s t r i k i n g differences i n the calculated V /K (25). T h e calculated V / K for B D oxidation i n B 6 C 3 F 1 mouse l i v e r m i c r o somes was sixfold greater than for rat a n d h u m a n l i v e r m i c r o s o m e s , a n d about 5 0 % h i g h e r than M M l i v e r microsomes. max

m a x

m

m a x

m a x

m

m

Microsomal Metabolism of B M O . I n i t i a l rates of e n z y m i c h y d r o lysis of B M O i n l i v e r microsomes w e r e l i n e a r w i t h p r o t e i n content o v e r the range of 0 . 2 - 1 0 m g / m L a n d w i t h t i m e for u p to 30 m i n (data not shown). E n z y m e - m e d i a t e d hydrolysis of B M O was not d e t e c t e d i n cytosolic fractions of l i v e r from any of the species. A l l 12 of the h u m a n l i v e r samples w e r e assessed for t h e i r ability to metabolize B M O u s i n g one i n i t i a l B M O c o n c e n tration (100 p p m ) . T h e n o r m a l i z e d first-order rate constants for the 12 l i v e r samples, w h i c h v a r i e d b e t w e e n 0.020 a n d 0.068 m i n m g p r o t e i n , w e r e t h e n r a n k - o r d e r e d , a n d three samples (high, m e d i a n , a n d l o w activity) w e r e selected for d e t a i l e d studies of B M O kinetics (see f u r t h e r on). I n a l l cases, - 1

Table II.

- 1

Kinetic Constants for the Oxidation of B D to Butadiene Monoepoxide"

Liver Microsomes Humans B 6 C 3 F 1 mice Sprague-Dawley MutaMouse

rats

max

(nmol/mg protein/min) 0.005 0.002 0.004 0.002

1.2 ± 0 . 6 2.6 ± 0.06 0.6 ± 0 . 1 1.7 ± 0 . 1

± ± ± ±

0.003 0.0002 0.0002 0.0002

"Values are mean ± standard deviation derived from model simulations (19). ^Values are in units of L*nmol*(min*mg protein* mmol) . -1


230 1295 157 850


148

results o f m o d e l simulations (19) adequately d e s c r i b e d the d e c l i n e i n B M O concentration i n the headspace d u e to e n z y m i c h y d r o l y s i s (data not shown). T a b l e I I I shows the M i c h a e l i s - M e n t e n constants ( K , V M

m a x

) for B M O

h y d r o l y s i s b y h u m a n , rat, a n d m o u s e ( B 6 C 3 F 1 a n d M M ) l i v e r microsomes. I n the t h r e e h u m a n l i v e r samples u s e d for the d e t a i l e d k i n e t i c e x p e r i m e n t s , V

m a x

r a n g e d from 9 to 60 n m o l / m g p r o t e i n / m i n , a n d apparent K s r a n g e d M


f r o m 0.2 to 1.6 m M , I n contrast, V

m a x

d e t e r m i n e d from reactions w i t h r o d e n t

l i v e r microsomes was o n e - h a l f or less of that m e a s u r e d i n h u m a n l i v e r s a m ples. Interestingly, the V

m a x

i n B 6 C 3 F 1 m i c e l i v e r microsomes for B M O h y -

drolysis was about fivefold h i g h e r t h a n i n M M l i v e r m i c r o s o m e s . A p p a r e n t K s for B 6 C 3 F 1 m i c e w e r e a p p r o x i m a t e l y s e v e n - to eightfold h i g h e r t h a n M

the K s o b s e r v e d i n rats a n d five times h i g h e r t h a n M M . A c o m p a r i s o n o f M

the calculated V

m a x

/K

M

reveals some s t r i k i n g differences across species (Table

III) . F o r e x a m p l e , for the three h u m a n l i v e r samples, calculated V ranged f r o m 32 to 38, whereas i n rodents, calculated V

m a x

m a x

/K s

/ K s ranged

M

from

M

4 to 10.

Conjugation of BMO with G S H ,

E n z y m e - m e d i a t e d conjugation o f

G S H w i t h B M O i n h u m a n a n d r o d e n t l i v e r cytosol c o u l d best b e d e s c r i b e d b y M i c h a e l i s - M e n t e n kinetics (Table I V ) . T h e V

m a x

i n B 6 C 3 F 1 mouse liver

cytosolic fractions was about twofold h i g h e r t h a n i n rat l i v e r cytosol (Table IV) . C o n j u g a t i o n of B M O w i t h G S H i n M M l i v e r cytosol was not m e a s u r e d i n these e x p e r i m e n t s . O n l y one of the two h u m a n l i v e r samples a n a l y z e d d i s p l a y e d M i c h a e l i s - M e n t e n kinetics a n d h a d a V

m a x

of one-fifth to o n e - t e n t h

of that o b s e r v e d i n rodents. M o u s e l i v e r cytosolic apparent K s w e r e about M

threefold h i g h e r t h a n that o f rats a n d h u m a n s . T h e c a l c u l a t e d V

m a x

/K

rodent l i v e r cytosol was about fourfold h i g h e r t h a n the c a l c u l a t e d V

in

M

m a x

/K

M

for the one h u m a n sample.

Discussion.

A n u n d e r s t a n d i n g o f the m e c h a n i s m s b y w h i c h B D i n -

duces tumors i n rats a n d m i c e is essential for extrapolating to h u m a n s , a Table III.

Kinetic Constants for the Hydrolysis of Butadiene Monoepoxide

a

V T

Liver Microsomes Humans

(high) (median) (low) B 6 C 3 F 1 mice Sprague-Dawley rats MutaMouse 6

(nmol/mg 58.1 18.5 9.2 5.8 2.5 1.1

K (mM) M

max

protein/min) ± ± ± ± ± ±

1.65 0.58 0.24 1.59 0.26 0.29

4.0 4.1 2.2 0.3 0.05 0.3

± ± ± ± ± ±

V /^M ma3

0.12 0.15 0.10 0.03 0.01 0.10

C

35 32 38 3.6 9.5 3.8

"Values are mean ± standard deviation derived from model simulations (19). ^Three of the 12 human liver samples were used for the kinetic analyses. Selection of the samples were as described in the materials and methods section. Values are in units of nmol-L-(min-mg protein-mmol)" . c

1


11.

BONDETAL. Table IV.


Kinetic Constants for Conjugation of Butadiene Monoepoxide with Glutathione" V

Liver Cytosol


149

Chemicals

Humans'' B 6 C 3 F 1 mice Sprague-Dawley rats MutaMouse

(nmol/mg

KM

max

(mM)

protein/min)

45.1 ± 5.8 500 ± 64 241 ± 3

^mJK

10.4 ±

1.04

35.3 ± 13.8 ±

6.2 0.3

4.3 14 17

d

d

C

U

d

"Values are mean ± standard deviation (SD) derived from model simulations. 'Two of the 12 human liver samples were used for kinetic analyses. Selection of the samples was as described in the materials and methods section. One of the samples displayed Michaelis-Menten kinetics and in the other sample, the reaction was best described by a rate constant of (2.56 ± 0.22) X 10~ L/mmol/min/mg protein (mean ± SD). 'Values are in units of L-nmol-(min-mg protein-mmol) . Not measured. 4

-1

rf

species for w h i c h B D carcinogenic p o t e n c y is at present u n k n o w n . It is l i k e l y that one of the c r i t i c a l b i o c h e m i c a l d e t e r m i n a n t s of B D - i n d u c e d carcinogen i c i t y is the extent to w h i c h B D is activated to epoxide metabolites that can react w i t h D N A to u l t i m a t e l y i n d u c e mutations. T h e i n d u c t i o n of mutations i n tissues of a transgenic m o u s e is a n o v e l approach to d e t e r m i n e i n v i v o m u t a t i o n i n d u c t i o n f o l l o w i n g exposure to carcinogens. T h e i n d u c t i o n of m u tations can be s t u d i e d w i t h i n the context of the endogenous p h a r m a c o k i n e t i c a n d biotransformation processes that d e t e r m i n e the tissue levels of reactive metabolites that can interact w i t h a n d alter the c e l l u l a r D N A i n various tissues. B D is a mutagenic carcinogen that exhibits species differences i n susc e p t i b i l i t y ; m i c e are m o r e susceptible to the genotoxic a n d carcinogenic effects of B D than are rats. Studies o n the i n v i t r o a n d i n v i v o m e t a b o l i s m of B D indicate that m i c e p r o d u c e m o r e B M O a n d B D E t h a n do rats (19,

26-

28). T h e s e data indicate that the biotransformation of B D to two reactive metabolites, B M O a n d B D E , exhibits species differences a n d is l i k e l y a c r i t ical d e t e r m i n a n t of the genotoxicity a n d c a r c i n o g e n i c i t y of B D . T h e relative balance b e t w e e n the i n v i v o bioactivation a n d detoxification pathways of B D d e t e r m i n e s tissue concentrations

of the u l t i m a t e m u t a g e n i c species,

the

quantities that interact w i t h D N A , a n d u l t i m a t e l y the type a n d a m o u n t of p r o m u t a g e n i c lesions i n the D N A of exposed animals. Because i n v i t r o systems cannot m i m i c c e r t a i n of these i n v i v o processes, it is essential that methods for the i n v i v o evaluation of m u t a g e n i c i t y be c o n s i d e r e d . E x p o s u r e of the M M strain of transgenic mouse to 625 p p m of B D for 5 consecutive days (6 h/day) f o l l o w e d b y a 14-day expression p e r i o d d i d not result i n significant m u t a g e n i c i t y i n l i v e r a n d b o n e marrow. H o w e v e r , s i g nificant m u t a g e n i c i t y was o b s e r v e d i n the l u n g , a target organ for the carc i n o g e n i c effect of B D i n B 6 C 3 F 1 m i c e at 6.25 p p m . Specific m u t a t i o n of the K-ras oncogene has also b e e n d e t e c t e d i n l u n g adenocarcinomas i n B D -



150


exposed m i c e (II). Because no carcinogenicity data i n the M M strain of mouse exist as they do for the B 6 C 3 F 1 mouse, it is difficult, at present, to relate the r e l a t i v e l y s m a l l increases i n m u t a g e n i c i t y o b s e r v e d i n the present studies to another biological e n d p o i n t (e.g., t u m o r formation). Studies are i n progress to evaluate the m u t a g e n i c i t y of B D u s i n g a B 6 C 3 F 1 mouse m u tation system (29). E x p o s u r e of B 6 C 3 F 1 m i c e to B D r e s u l t e d i n significant genotoxicity as d e t e r m i n e d b y m i c r o n u c l e u s ( M N ) , sister c h r o m a t i d exchanges ( S C E ) , and c h r o m o s o m a l aberration ( C A ) analysis (15,16). S i g n i f i cant levels of M N (sixfold above control levels), a n d S C E w e r e o b s e r v e d i n B 6 C 3 F 1 m i c e f o l l o w i n g 2-day exposures (6 h/day; nose only) at levels of 100 p p m B D a n d h i g h e r (16). In the same study, exposure of rats to 100-10,000 p p m (6 h/day for 2 days) d i d not result i n significant increases i n S C E or M N . E x p o s u r e of B 6 C 3 F 1 m i c e to 6.25, 62.5, a n d 625 p p m B D 6 h/day for 10 days r e s u l t e d i n a significant d o s e - d e p e n d e n t increase i n S C E (at 6.25 p p m ) , M N (at 62.5 p p m ) , a n d C A (at 625 p p m ) (15). T h e s e data o n the c y togenetic effects of B D i n rats versus m i c e are consistent w i t h studies of the biotransformation of B D . T h e r e f o r e , k n o w i n g h o w mouse strain differences i n B D biotransformation w i l l affect the p o t e n t i a l genotoxicity of B D must await p a r a l l e l m u t a t i o n studies w i t h B 6 C 3 F 1 transgenic m i c e (29) a n d cytogenetic studies i n M M . T h e data p r e s e n t e d i n this chapter reveal that significant species differences i n the V is o b s e r v e d for B D oxidation to B M O (see Table II). F o r example, B 6 C 3 F 1 m o u s e l i v e r microsomes d i s p l a y e d a capacity for B D o x i dation exceeding that seen i n e i t h e r h u m a n or rat liver. T h i s capacity was e v i d e n c e d b y a c o m p a r i s o n of b o t h the m a x i m u m velocity for the reaction, V , and V / K . T w o putative detoxification e n z y m i c reactions can occur w i t h B M O , hydrolysis b y epoxide hydrolase a n d conjugation w i t h G S H b y glutathione transferase. T h e results from o u r studies reveal that l i v e r tissues from a l l species can detoxify B M O b y b o t h pathways (Tables III a n d I V ) . I n general, h u m a n l i v e r microsomes h y d r o l y z e d B M O at greater rates than those of e i t h e r rats or m i c e , as e v i d e n c e d b y the h i g h e r V and V /K . A l t h o u g h there was a considerable range of epoxide hydrolase activities i n l i v e r microsomes of the three h u m a n samples investigated ( V = 9-58 n m o l / m g p r o t e i n / m i n ) , values for the h u m a n samples w e r e at least twofold greater than the V for rats a n d m i c e . T h e value r e p o r t e d b y K r e u z e r et al. (30) for epoxide hydrolase-catalyzed h y d r o l y s i s of B M O b y microsomes from a single h u m a n l i v e r sample ( V = 14 n m o l / m g p r o t e i n / m i n ) falls w i t h i n the range of values for V i n h u m a n l i v e r microsomes r e p o r t e d i n this chapter. Values for V / K r e p o r t e d b y K r e u z e r et a l . (30) for rodents a n d the one h u m a n l i v e r sample w e r e s i m i l a r to the values r e p o r t e d i n this chapter. F o r a l l species, apparent K s for h y d r o l y s i s a n d G S H conjugation (not assessed i n M M ) w e r e significantly greater than for B D oxidation a n d , i n the case of m i c e , B M O oxidation. m a x

m a x

m a x

M

m a x

m a x

M

m a x

m a x

m a x

m a x

m a x

M

M

Studies o n the biotransformation of B D b y M M l i v e r microsomes i n d i cate that M M enzymes catalyze the conversion of B D to B M O a n d h y d r o -


11.

BOND ET AL.


Chemicals

lysis o f B M O . A comparison o f the initial rates ( V / K ) of B D m e t a b o l i s m to B M O a n d B M O hydrolysis b e t w e e n M M a n d B 6 C 3 F 1 mouse l i v e r m i crosomes indicates that there are also mouse strain differences i n B D m e tabolism. T h e ratio of V / K for B D to B M O and B M O hydrolysis i n M M l i v e r microsomes was 225:1, whereas t h e same ratio i n B 6 C 3 F 1 mouse l i v e r microsomes is 360:1. T h e lack o f m u t a g e n i c i t y o f B D i n t h e l i v e r of the M M may b e d u e to a n ineffective concentration of B M O i n t h e liver. D e t a i l e d parallel studies o n the biotransformation o f B D , i n v i t r o a n d i n v i v o , i n B 6 C 3 F 1 and M M are r e q u i r e d to establish t h e role o f B D bioactivation a n d detoxification i n m e d i a t i n g the genotoxic effects of B D . m a x

m a x


151

M

M

T h e interaction o f chemicals o r t h e i r metabolites w i t h D N A is a major factor i n c h e m i c a l carcinogenesis. O n e p o t e n t i a l strategy for m o r e accurately estimating h u m a n h e a l t h risks from exposure to D N A - r e a c t i v e chemicals is to d e v e l o p research that w i l l p r o v i d e a n d i m p r o v e u n d e r s t a n d i n g o f the mechanisms of action of chemicals w i t h i n a n exposure —> tissue dose —» c a n cer response p a r a d i g m . T h e d e v e l o p m e n t o f quantitative linkages b e t w e e n exposure a n d response, w h i c h are based o n biologically p l a u s i b l e m e c h a nisms o f action at exposure levels that are l i k e l y to b e e n c o u n t e r e d b y p e o p l e , w i l l significantly i m p r o v e the risk assessments for h u m a n exposures to D N A - r e a c t i v e chemicals. T h i s study was i n i t i a t e d to b e g i n to establish i n v i v o linkages b e t w e e n exposure to B D , i n t e r n a l dose, a n d a biological r e sponse (mutation). T h e i n d u c t i o n of mutations is a d e t e r m i n a n t o f B D i n d u c e d carcinogenicity. A l t h o u g h these studies d i d not investigate t h e carcinogenicity of B D i n M M , t h e i n d u c t i o n o f mutations i n a transgene integrated into t h e g e n o m e o f t h e M M represents a n i n i t i a l step t o w a r d establishing d o s e - r e s p o n s e relationships for m o l e c u l a r events (mutation) that are part o f the carcinogenic process. T h e s e relationships n e e d to b e established w i t h endogenous genes that are k n o w n to b e i n v o l v e d i n the carcinogenic process (e.g., oncogenes a n d t u m o r suppressor genes). T h e i n vitro metabolic constants from these studies c a n b e i n c o r p o r a t e d into p h y s iological models that can simulate i n v i v o behavior, a n d t h e models c a n b e used to p r e d i c t b l o o d a n d tissue concentrations of B D a n d B M O . E x p e r i ments u s i n g w h o l e animals can t h e n b e u s e d to verify m o d e l p r e d i c t i o n s based o n i n v i t r o - d e r i v e d rates. U l t i m a t e l y , i n v i t r o - d e r i v e d rates for h u m a n tissue can t h e n b e used to p r e d i c t B D a n d B M O concentrations i n h u m a n tissues as a first step i n e s t i m a t i n g risk d u e to B D exposure.

Acknowledgments T h e authors gratefully acknowledge t h e n u m e r o u s valuable discussions w i t h a n u m b e r o f o u r colleagues.

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RECEIVED

for r e v i e w

September 3 ,

1992.

ACCEPTED

revised

Toxicol.

manuscript

J a n u a r y 26, 1993.


Environmental Epidemiology - American Chemical Society

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