Fluroxene mutagenicity

183

Mutation Research,

58 (1978) 183--191 © Elsewer/North-Holland Biomedmal Press

F L U R O X E N E MUTAGENICITY

J E F F R E Y M. B A D E N * , M E R I J E A N K E L L E Y * *, V I N C E N T F . S I M M O N * * *, S U S A N A . R I C E * * a n d R I C H A R D I. M A Z Z E * * * *

Department of Anesthesm, Stanford Unwerszty School of Medzcme, Stanford, Calif. and Anestheszology Servzce, Veterans Adminzstratmn Hospztal, Palo Alto, Calif. and Department of Toxtcology, Stanford Research Instztute, Menlo Park, Cahf. (U.S A.) (Received 10 February 1978) (Revismn received 28 April 1978) (Accepted 15 May 1978)

Summary The commercially available volatile anesthetic fluroxene (2,2,2-trifluoroethyl vinyl ether) which contains the stabilizer N-phenyl-l-napthylamine, was tested for mutagenicity usmg four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100, and one strain of E. coli, WP2. In addition, purified fluroxene; N-phenyl-l-napthylamine; trifluoroethanol, a major metabolite of fluoroxene; and urine from rats anesthetized with fluroxene were tested. Several procedures were utilized including exposure of bacteria to vapor in desiccators and in liquid suspension. Results indicate that fluroxene, b u t not its stabilizer, was mutagenic to strains TA1535, T A 1 0 0 and WP2 only in liquid suspension and only in the presence of a rat-liver enzyme system. Trifluoroethanol and urine from fluroxene-treated rat were not mutagenic to any strain of bacteria. These findings indicate that fluroxene is a promutagen which requires preincubation before it is recognized. Further experiments were performed with enzymes prepared from mouse, hamster and human liver. Fluroxene was mutagenic only in the presence of enzymes prepared from Aroclor 1254 pretreated rodents. Since fluroxene was n o t mutagenic in the presence of enzymes prepared from three human livers, the significance of these findings to man are unclear.

* Asslstant Professor of Anestheszology. ** Research Assoclate in Anestheslology. *** Manager, M1croblal Genetlcs Program, Stanford Research I n s h t u t e . * * ** Professor of Anestheslology. Supported In part by the VA Hospltal, Palo Alto, Cahf. Presented at the 1977 A n n u a l Meetlng of the American Soclety of Anestheslologlsts, New Orleans, La (U.S A )

184 Introduction

Recent epldemlologic surveys suggest an Increased incidence of malignancies in operating room personnel [8,9]. A possible cause is chronic exposure to trace concentrations of anesthetm gases present in the operating suite. To determine if such carcmogenlc potentml exists, we have previously examined several commonly used volatile anesthetms [3,4] in the Salmonella/mammalian enzyme system developed by Ames and coworkers [1,2]. This assay is useful in detecting carcmogens as mutagens [16]. We reported that only the anesthetic, fluroxene (2,2,2-tnfluoroethyl vinyl ether) m liquid suspension and m the presence of metabolic system, was mutagenic [4]. The fluroxene tested was the commercially available preparatmn which contained a nonvolatile stabilizer, N-phenyl-l-naphthylamine (0.01% w/w). It was uncertmn whether fluroxene or its stabilizer (or both) was the mutagen; these experiments have been designed to resolve that issue. Furthermore, the metabolism of fluroxene is qualitatively different in rodents and man [7,10]. To better assess the possible hazard of this agent, experiments were performed using enzymes prepared from mouse, hamster and human hver. Trlfluoroethanol, a major metabolite of fluroxene in rodent, but not in man, was also tested. Finally, an attempt was made to detect mutagenic components m the urine of rats anesthetized with fluroxene. Materials and methods Chemicals Fluroxene was obtmned from Ohio Medmal Products (Madison, Wls.) and further purified by low temperature vacuum distillation. Purified fluroxene (2,2,2-trifluoroethyl vinyl ether) was confirmed to be 99.9% pure by gas chromatography and m particular contained no trace (<1 ppm) of the stabilizer N-phenyl-l-naphthylamme. N-Phenyl-l-naphthylamine was obtmned from Ohio Medical Products; trifluoroethanol from Aldrich Chemical Company (Milwaukee, Wis.); vlnylidene chloride from D u p o n t (Wilmington, Del.); 2-anthramine from Pfaltz and Bauer, Inc. (Flushing, N.Y.). Bacterial strains Histydlne-dependent tester strains of S. typhimurium, (TA1535, TA1537, TA98 and TA100) and a tryptophan-dependent strain of E. coh (WP2), were employed as mdlcators of mutagenicity [6,16]. For each experiment inocula from stock cultures were grown overnight at 37°C in nutrient broth and used the next morning. Mammahan metabolzc systems R o d e n t 9000 × g supernatants (S-9) were prepared from livers of male Fischer 344 rats, Syrian golden hamsters and B6C3F1 hybrid mice. S-9 was prepared both from untreated and polychlonnated biphenyl-(Aroclor 1254} treated animals. Immediately prior to use, S-9 mix was prepared as follows: an ahquot of S-9 was thawed and 2.0 ml was added to 0.05 ml, 1 M glucose6-phosphate; 0.2 ml, 1.65 M KC1; 0.2 ml, 0.4 M MgC12; 0.4 ml, 0.1 M NADP;

185 5.0 ml, 0.2 M sodium phosphate (pH 7.4); and sterile water. The final volume was 10 ml. A second mixture to be used as a control, was similarly prepared but w i t h o u t the S-9 and NADP. Human hver. Liver was obtained from three prewously healthy male hearttransplant donors who had suffered irreversible brain damage following head injuries. Each patient's pulse and blood pressure were mmntained at physiological levels from the time of hospital admission until cardiectomy. Livers were removed within 1 h of cardmctomy. The preparation of a S-9 from each liver was identical to that used for rat liver. The a m o u n t of protein m the S-9 prepared from each specms was measured and standardized to 50 mg/ml [14].

Mutatton tests 50 gl of overmght culture of Salmonella (~109 organism/ml). 2 ml of molten top agar containing 0.1 pM histidine, and either 0.5 ml of metabolic system or control mixture were mixed and plated on glucose minimal medium. For the E. coli strain, WP2, no histidine was added to the top agar and the mixture was plated on 0.125% oxoid medium [6]. Triplicate plates were prepared at fluroxene and tnfluoroethanol vapor concentrations ranging from 0.1 to 30% and at N-phenyl-l-naphthylamine concentrations ranging from 0.01--1000 pg plate; each experiment was performed at least twice. Vinylidme chloride (3%) [5] and 2-anthramine (2.5 gg/plate) [16] were used as positive controls, since they require active S-9 before they are recognized as mutagens. In an initial series of desiccator and hquid incubation experiments, vapor concentrations of fluroxene were verifmd by gas chromatography and were f o u n d not to vary from predicted values by more than 10% nor to vary significantly with time. These procedures have been described m detail [3,16]. The following is a hst of the tests conducted and a brief description. (1) Desiccator incubation experiments. Plates were incubated for 8 h at 37°C m ~ev]ed desmcators contmnmg the above vapor concentrations of commercial fluroxene. Plates were then removed from the desiccators and incubated for an additional 40 h. In other desmcator experiments, bacterial stratus TA1535, TA100 or WP2, were exposed to fluroxene vapor concentrations of 10 or 30% for 2, 8, 12, 24 and 48 h. (2) Liquid suspenston experiments. Bacteria with either metabolic system or buffer were exposed to either p u n f m d fluroxene, commercial fluroxene, Nphenyl-l-naphthylamine or trifluoroethanol m sealed tubes for 2 h at 37°C. Molten top agar was then added to each tube and the contents were plated. In several expenments, surwval of the bacteria was determined by dflutmg an aliquot of the suspensmn mixture 106 fold before plating. Plates were incubated at 37°C for 48 h. (3) D~rect plate assay. Bacteria, N-phenyl-l-naphthylamine, top agar, and metabohc system or buffer, were mixed, plated immedmtely and incubated for 2 days at 37°C. (4) Urine assay. 24-h urme samples were collected from each of 6 rats, prior to and on days 1, 2, 3 and 7 after 1-h exposure to 4% fluroxene. 3 of the rats had received phenobarbital in their drinking water (approximately 30 mg/day) for 7 days prior to fluroxene anesthesm [15]. Each u n n e sample was passed through a 0.22-p pore size membrane filter (Mfllipore, Bedford, Mass.) and

186

0.25 ml of filtrate was combined with bacteria in top agar with or without the metabolic system, and with or w i t h o u t ~-glucuronidase (650 units/sample). This mixture was plated immediately and incubated for 48 h at 37°C before being counted.

Analysis of data The numbers of revertant colonies on treated plates were compared with the numbers of spontaneous revertant colonies on plates exposed to room air. Student's t-test was used for statistical analysis, P < 0.05 was considered significant. Results

In desiccators, exposure for 8 h to fluroxene at vapor concentrations ranging from 0.1 to 30% was not mutagenm to any of the tester strains, with or without the rat S-9 mix. Results of desiccator experiments in which strains TA1535, TA100 and WP2 were exposed for 2, 8, 12, 24, or 48 h to 10 or 30% fluroxene vapor concentration, were also negatwe. In the liquid suspension assay, however, commercial fluroxene was mutagemc to stratus TA1535, TA100 (Fig. 1) and WP2, in a dose-dependent manner, at vapor concentrations between 3 and 30% but only m the presence of the S-9 mix. There was no mutagenm effect on strains TA1537 and TA98. Survwal of bacteria m these preincubation studies was reduced approximately 50% at 30% fluroxene vapor concentration. A typical survival curve for TA1535 is shown m Fig. 2. The fluroxene metabohte trifluoroethanol was not mutagenic to any tester strain when assayed m hquid suspension. Purified fluroxene containmg no N-phenyl-

300 -

250 ~. = With S-9 Mix o- - --.oWbthout S-9 Mix

200

a

150 E

rr

100

50

o

¢/, 0 1

I

0 2

. . . .

014 0'6

I

I

I

1

2

4

'

;

6

'

'

'

1'0

2'0

4'0

Percent F l u r o x e n e Fig 1 Number of revertant colonies per plate of S typhtmur~um, TAIO0, after hqmd suspension with c o m m e r c i a l f l u r o x e n e , w i t h o r w i t h o u t S-9 m i x . F l u r o x e n e i s m u t a g e m c a t v a p o r c o n c e n t r a t i o n s g r e a t e r t h a n 3%.

187 100 60. 40-

20.

E

10-

;

--" With S-9 Mix

10

20

30

\

40

50

60

Percent Fluroxene F i g . 2. P e r c e n t s u r v i v a l o f S, typhzmurzum, T A 1 5 3 5 , a f t e r h q m d s u s p e n s i o n w i t h c o m m e r c i a l f l u r o x e n e , w~th o r w i t h o u t S-9 mLx. F l u r o x e n e is t o x i n at v a p o r c o n c e n t r a t t o n s g r e a t e r t h a n 2 0 %

1-naphthylamine was mutagenm to the same bacterial strains at similar concentrations. In contrast, the stabilizer, N-phenyl-l-naphthylamine tested in direct plate and liqmd incubation assays was n o t mutagenic to any of the tester strains in amounts up to 1 0 0 0 pg/plate (Fig. 3). This quantity is 200 times more than the amount of N-phenyl-l-naphthylamine present in the highest concentration of fluroxene found to be mutagenic. Fluroxene was mutagenic

3u-

25-'

E.

20" -,o

15E

rr

= : With S-9 Mix c- - -.o Without S-9 Mix

10-

5-

o- -I I , 01

02~ ' 0~4 "016 ' ' 11

i

2

'

i ' i'"J

4

6

10

2

I0

' 4

0

'6 I'''l

0 100

i

200

,

i

400

.....

i

1000

N-phenyl-l-naphthylamme (,ug/plate) F i g 3. N u m b e r o f r e v e z t a n t c o l o m e s p e r p l a t e o f S t y p h z m u n u m , T A 1 5 3 5 , i n a d t r e c t p l a t e a s s a y w i t h N - p h e n y l - l - n a p h t h y l a m m e , w i t h or w i t h o u t S-9 m i x . N - P h e n y l - l - n a p h t h y l a m m e Is n o t m u t a g e m c a t a n y o f the concentrations tested.

188 TABLE

1

FLUROXENE Agent

MUTAGENICITY, Concentrahon

S-9 PREPARED

1% 5% 10%

OF DIFFERENT

TA100 I d

0 10 --3

TA1535

U

I

TA100

U

5 35 e 93 e

2 1 10

--5 26 e 152 e

I

U

--23 -207 e

9 21 93 16

38 e

--

269 e

13

75 e

--9

148 e

1

105 e

3%

--5

88 e

16

126 e

_

_

2.5 pg/

105 e

548 e

114 e

2219 e

I

--25 5 7

30% Vmyhdene

SPECIES a

Mouse

U c

chloride 2-Anthramme

LIVERS

Rat TA1535

Fluroxene

FROM

1228 e

349 e

3 63 e 131 e

1121 e

1254 e

plate a % increase of revertant colomes above control b Numbers refer to mchmdual human hvers c U, Livers prepared d I, L i v e r s p r e p a r e d

from umnduced

ammals

from ammals treated

e Greater than room

5 days prior to sacnhce

within 500 mg]kg Aroclor 1254

atr control p ~ 0 01

m liquid suspension assay m the presence of S-9 mix prepared from Aroclor 1254 induced rat, mouse and hamster liver, bu t n o t in the presence of S-9 mix prepared from h u m a n liver or from u n m d u c e d rat, mouse or hamster liver (Table 1). Assays with T A 1 5 3 5 or T A 1 0 0 on the unnes of fluroxene anesthetized rats showed no increase in the n u m b e r of revertant colomes (Table 2).

TABLE

2

URINARY

MUTAGENESIS Strata

S-9

EXPERIMENT

NUMBER

Glucurom-

Preanes-

dase

thetlc

OF REVERTANT Postanesthetac 1

Untreated

TA1535

TA100

Phenobar-

TA1535

bital treated rats TA100

--

52

+

9

42-+

--

+

48

+

9

47

+ +

-+

1 9 +_ 4 24 ± 4

--

--

127_+

6

--

+

135

6

+ +

-+

1 4 4 +- 9 146 ± 9

--

--

35

±

7

54

--

+

40

±

8

+ +

-+

31 ± 3 9 -+

5 8

44-+ 24+48-+

8 6 6

--

--

133

t 11

128 ±

7

--

+

145±

+ +

-+

172 + 14 155 + 11

9

MEAN

-+ S E

(day)

2

--

+

COLONIES,

3

7

1

51+

7

45±

8

46_+

8

+

8

42±

7

43

+

9

9

21 + 18+

5 2

36 + 17 ±

5 2

27 ± 14 +

5 3

50-+ 26_+ 24 +

124_+ 13 1 3 9 _+ 1 3 140 ± I0 159 + 10 + 11

10 140 + 13 1 3 1 -+ 6 130

t

142_+ 12 1 2 4 +_ 8 148 + 8 147 + 9 51 50 29 26

+ 10

144 151 138 148

9

+

9

-+ I I -+ 9

47 + 44-+ 40--27-+

±

7

± ±

9 6

+

7

166

1 4 8 • 10 1 6 5 -+ 1 2 162 ± 13

159

148

+

+ 13

8

55

39 + 2 4 +3 3 -+

13 174 + 13 1 4 1 -+ 6 t

138_+ 12 160 + 14 153 + 9 164 + 12

9 7 6

• 19

5 4

7

7 5

150-+ 16 146-+ 7 139 + 9 176 ± 17

189

Man b

Hamster TA1535

TA100

U

U

I

--15 .

--10 .

2200 e

. 2580 e

I

--25

. 20 e 30 e

7 5 .

TA1535 1

.

94 e 162 e .

221 e

2

19

. 8 12

TA100

. 1422 e

3

1

2

3

13

--8

12

10

--15

--12

7 --25 --6

0 --20 0

0 8 --16

5 --17 14

--20 --25 --15

2 --8 --2

. 1880 e

. 384 e

2144 e

66 e

111 e

_

418 e

186 e

963 e

Discussion

The volatile fluorinated anesthetic fluroxene, has been shown to be mutagenic to S. typhlmurium strains TA1535 and TA100 in a liquid suspension assay [4]. The present study confirms this fmdmg and in addition shows that fluroxene is mutagenic to E. coh, WP2. Commercial fluroxene contains a small amount (0.01% w/w) of the stabilizer N-phenyl-l-naphthylamine, a chemical that is structurally similar to the bladder carcinogen 2-naphthylamine [13]. One would predmt that, if mutagenic, the simple aliphatic molecule fluroxene would be a base-pmr mutagen and the cychc aromatic amine, Nphenyl-l-naphthylamine, would be a frame-shift mutagen. In fact, the commercial fluroxene mixture contmning N-phenyl-l-naphthylamine, was mutagenic only to the bacterial strains most susceptible to base-pair mutation (WP2, T A 1 5 3 5 and TA100) and not to those strains sensitive to frame-shift (TA1537, TA98). These experimental findings are consistent with fluroxene being a promutagen. Furthermore, N-phenyl-l-naphthylamine was n o t mutagenic under any of the conditions tested, whereas purified fluroxene containing no N-phenyl-l-naphthylamme, was as mutagenlc as the commercial preparation. A surprising finding of this study is that fluroxene was mutagenic only m the hquid assay. It was n o t mutagenm to any tester strata exposed m desiccators even at exposure times up to 48 h. It has been reported that preincubation or suspension assays are required to detect the mutagenic activity of some nitrosamines and aromatm alkylamines [17]. The most likely explanation for this p h e n o m e n o n is that preincubation results in an increased concentration of mutagenm metabolites. Whatever the reason, our findings identify a class of promutagens which reqmre preincubation to be recognized. Less unusual, was the fmdmg that fluroxene was mutagenm only in the presence of S-9 mixes prepared from Aroclor 1254 pretreated rodents. This

190 pretreatment induces the mixed function oxldase system and thereby qualitatively and quantitatively alters a drug's biotransformation. Fluroxene was n o t mutagenic in the presence of human S-9 preparations. In this respect the livers of previously healthy heart donors, not known to have been taking any medications, resembled those of the non-Aroclor1254 treated rodents. Support for this observation comes from other m vitro studies in whmh these human-liver preparations were used to defluorinate several volatile anesthetics; actwity was equal to or less than that obtmned with non-induced rat liver* [11]. However, these simllaritms should be vmwed with caution for the metabolic activity of the human hver may n o t have been completely normal. Specifically, the h u m a n hvers were obtamed from heart donors whose normal physiologm conditions were maintained by artlflcal means, and a delay of 30-60 min occurred from the time of cardiectomy until the liver could be removed and cooled. Furthermore, livers from only three human subjects were used in this experiment. This number is too small to be considered representative of the human population as a whole. In spite of these considerations the data obtmned with a human metabohc system is useful since the metabohsm and toxmlty of fluroxene m man may differ from other animal species. For example, m the mouse, fluroxene is biotransformed to trifluoroethanol. The toxicity of this metabohte may be responsible for the observation that mice exposed for 4 h to 4.5% fluroxene died within 24 h [7]. Interestingly, however, trifluoroethanol was n o t mutagenic under the conditmns tested. Rats are also subject to toxin effects form fluroxene exposure especially ff liver enzymes have been prewously induced with phenobarbital [10]. In our study 6 rats were anesthetized for 1 h with 4% fluroxene. Of the 3 animals treated with phenobarbital, one dmd shortly after fluroxene exposure and the other two recovered more slowly than the untreated animals. In man, fluroxene is pnmflanly biotransformed to trifluoroacetm acid w i t h o u t serious toxin effect [12]. Species varlatmns in drug metabohsm and toxicity emphasize the difficulty extrapolating results from one specms to another and from in vitro experiments to in vivo situations m man. Fluroxene has been used as an anesthetm smce 1954, although its productmn was discontinued m 1975. It is the only anesthetm that has been found to be mutagenm m the bacterial mammalmn enzyme system. Smce it was n o t mutagenre m the presence of enzymes prepared from 3 human hvers, the slgmflcance of these findings to man are unclear. However, the possibility that fluroxene m a y have c o n t n b u t e d to the mcreased rate of malignancy reported to occur m operating room personnel cannot be excluded. References 1 A m e s , B N , T h e d e t e c t i o n of c h e m i c a l m u t a g e n s w i t h e n t e r i c b a c t e r i a , in A H o U a e n d e r ( E d ) . C h e m m a l M u t a g e n s Principles a n d M e t h o d s f o r T h e i r D e t e c t i o n , P l e n u m , N e w Y o r k , 1 9 7 1 , pp. 2 6 7 ~ 282 2 A m e s , B N , W E D u r s t o n , E Y a m a s a k i e t al., C a r c i n o g e n s are m u t a g e n s A s i m p l e test s y s t e m c o m bining h v e r h o m o g e n a t e s f o r a c t i v a t i o n a n d b a c t e r i a l f o r d e t e c t i o n , Proc. Natl. A c a d Scl. ( U S.A ). 70 ( 1 9 7 3 ) 2 2 8 1 - - 2 2 8 5

* U n p u b l i s h e d d a t a , S u s a n Rice, Ph D , D e p t o f A n e s t h e s i a , S t a n f o r d U m v e r s l t y C a h f o r m a

191

3 B a d e n , J . M , M B r l n k e n h o f f , R.S. W a h r t o n e t a l , M u t a g e m m t y o f volatile a n e s t h e t i c H a l a t h a n e , Anesthesiology, 45 (1976) 311--318. 4 B a d e n , J M., M. K e l l e y , R S. W h a r t o n et a l , M u t a g e m m t y o f h a l o g e n a t e d e t h e r a n e s t h e t i c s , A n e s t h e s i ology, 46 (1977) 346--350. 5 B a r t c h , H , C Malaveflle, E. Y a m a s a l u et a l , Tissue m e c h a t e d m u t a g e m m t y o f v m y h d e n e c h l o r i d e a n d 2-chlorobutachene m S typhlmurmm, Nature (London), 255 (1975) 641--643. 6 Bridges, B.A , S i m p l e b a c t e r i a l s y s t e m s f o r d e t e c t a n g m u t a g e m c agents, L a b P r a c t , 21 ( 1 9 7 2 ) 4 1 3 - 419 7 Caseorbl, H . F . , B l o t r a n s f o r m a t i o n o f d r u g s u s e d m a n e s t h e s i a , A n e s t h e s i o l o g y , 39 ( 1 9 7 3 ) 1 1 5 - - 1 2 5 8 C o h e n , E N., B W. B r o w n , D L B r u c e et al., O c c u p a t i o n a l chsease a m o n g o p e r a t i n g r o o m p e r s o n n e l A n a t i o n a l s t u d y , R e p o r t o f a n A d H o c C o m m i t t e e on t h e E f f e c t o f T r a c e A n e s t h e t i c s o n t h e H e a l t h o f O p e r a t i n g R o o m P e r s o n n e l , A m e n e a n S o m e t y of A n e s t h e s i o l o g i s t s , 41 ( 1 9 7 4 ) 3 2 1 - - 3 4 0 . 9 C o r b e t L T H , R G Cornell, K Llechng e t a l , I n m d e n c e o f c a n c e r a m o n g Michigan n u r s e - a n e s t h e t i c s , A n e s t h e s i o l o g y , 38 ( 1 9 7 3 ) 2 6 0 - - 2 6 3 10 H a r n s o n , G . G . , a n d J.S S m i t h , Massive l e t h a l h e p a t i c necrosis m rats a n e s t h e t i z e d w i t h f l u r o x e n e , a f t e r m l c r o s o m a l e n z y m e r e d u c t i o n , A n e s t h e s i o l o g y , 39 ( 1 9 7 3 ) 6 1 9 - - 6 2 5 11 H l t t , B A , R.I. Mazze, W.J. B e p p u et al • E n f l u r a n e m e t a b o h s m m rats a n d m a n , J P h a r m a c o l E x p T h e r , 203 (1977) 193--202. 12 J o h n s o n , R R , T . H C r o m w e l l , E g e r II et a l , T h e t o x i c i t y of f l u r o x e n e m anLmals a n d m a n , Anesthesiology, 38 (1973) 313--319 13 I A R C M o n o g r a p h o n t h e E v a l u a t i o n o f t h e C a r e m o g e m c Risk o f C h e m i c a l s t o Man, I A R C Scientific Pubhcatlons, WHO Lyon, 4 (1974) 97--111 14 L o w r y , O H , N J R o s e n b r o u g h , A L. F a r r a n d R J. R a n d a l l , P r o t e i n m e a s u r e m e n t w i t h t h e f o h n p h e n o l r e a g e n t , J Biol. C h e m , 193 ( 1 9 5 1 ) 2 6 5 - - 2 7 5 15 Marshall, W j , a n d A.E. M c L e a n , T h e e f f e c t o f oral p h e n o b a r b l t o n e on h e p a t i c m l c r o s o m a l c y t o c h r o m e P - 4 5 0 a n d d e m e t h y l a t m n a c t i v i t y in rats fed n o r m a l a n d l o w p r o t e i n diets, B l o c h e m . P h a r m a col ~ 18 ( 1 9 6 9 ) 1 5 3 - - 1 5 7 16 M c C a n n , J E , E ChoL E Y a m a s a l a e t al., d e t e c t i o n of c a r c i n o g e n s as m u t a g e n s m t h e S a m o n e l l a / m l c r o s o m e test P a r t I, A s s a y o f 3 0 0 c h e m i c a l s , Proc. Natl. A c a d ScL ( U S.A ), 72 ( 1 9 7 5 ) 5 1 3 5 - 5139 17 Yahagl, T , M D e g a w a , Y S e m o e t a l , M u t a g e m c l t y of c a r c m o g e m c azo d y e s a n d t h e i r d e n v a t a v e s , Cancer Letters, 1 (1975) 91--96.