Genotoxicity studies with bile conjugate metabolites

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acids as promoters of colon carcinogenesis (Reddy iit (11.. 1977; Wynder ... ('crrc,irio,~c.ri~,.\iv. 4. I I79 I I X3. Nt///. Ac~/c/. Sc'i. C'..S.d. 79. 5971 5975. Vol. 14.



the relative rate of labelling of phosphatidylglyccrol and DGDG while that for phosphatidylcholine decreased. In both algae, illumination increased the relative labelling of oleate and linoleate. W e a r e grateful to the S.E.R.C. (studentship to T . R . P . ) for linancial hupport. Anderson. R., Livermore. B. P., Katcs. M . & Volcani, H. E. (197X) B f f ) < h f f Bfr'/J/f\~.s. ff. .4c,/u 528. 77 XX Courchaine. A . J., Miller. W . f l . & Stein. D. B. (1959) ( ' / / t i . ( ' h c w i . 5 .

Genotoxicity studies with bile conjugate metabolites MARGARET H. BLAKEBOROUGH,* ROBERT W. OWENt and RODNEY F. BILTON * Dcpurtnirwt of Chcmistrj~und Biochemistry. Liwrpool Polj~tcdinic.Bjwlrvi Strcc>t, Liwrpool L3 3A F. U . K . , untl t P H L S Ccntrc j i ) r Applied Microbiology und Roscur ch . BNCt c>ri(ilM c t uholisni Rescur ch Lu horu t o r!-, Porron Down. Suli.shurj*,WiltshirP. SP4 OJG. U .K . There has been strong epidemiological evidence correlating dietary fat intake to the incidence of colorectal cancer (Armstrong & Doll. 1975). Faecal profiles of populations on high-fat diets show high levels of bile acid derivatives. total neutral sterols and anaerobic bacteria capable of actively metabolizing cholesterol and bile acids to possible carcinogens and/or co-carcinogens (Hill (it (11.. 1970; Reddy & Wynder, 1973). There is much evidence to date implicating certain bile acids as promoters of colon carcinogenesis (Reddy iit (11.. 1977; Wynder & Reddy. 1977). Much of this evidence is derived from animal studies which can be both longterm and expensive. We have therefore turned to microbial mutagenicity assays. particularly the Ames test (McKillop (11.. 1983). for testing bile conjugate metabolites produced by bacterial transformation. The process of steroid transformation by bacteria is time consuming and yields only small amounts of any one compound for testing. For this reason we chose a relatively new bacterial assay developed by Quillardet cr (11. ( 19x3) called the SOS-chromotest. This test is a simple and quick colorimetric assay based on the induction of the SOS function .!/;A, whose level of expression is monitored by means of a .!/;A : : IucZ operon intusion. 1 he response is rapid (a tew hours) and does not require survival of the tester strain Esihcriciri i d i PQ37. PQ37 is constitutive for alkaline phosphatase and produces /&galactosidase when DNA damage occurs. Compounds tested using the SOS-chromotest may at certain concentrations inhibit protein synthesis which would. in turn, lead to an underestimation of /jgalactosidase induction. To correct for this, general protein synthesis is estimated during the incubation period by simultaneously measuring alkaline phosphatase synthesis along with /I-galactosidase. The ratio of [I-galactosidase activity to alkaline phosphatase activity is taken as a measure of the specific activity of [j-galactosidase. This Abbreviations used: SOSIP. SOS-inducing potency; DMSO, dinicthyl sulphoxide.

Vol. 14

Ta hlc I . XIS-inciuc~in,qp o i ~ v i c ~ i co~fs'hik c~otrjrcgrriot n c , i d ~ o I i i ~ , ~ on(/ pcrriwi h i k clc.icl.c ('om pounds tested


(I )

Androsta-I .4.6-tricnc-3. I7-dioIlc in I l M S O ( 2 ) 3.5-Cholcstadienc in DMSO ( 3 ) Cholcstcrol-5z-6z-ep(~xidcin D M S O ( 4 ) 12~-~~ydroxychol~i-4.h-dicne-3-onc-24-oic acid i n D M S O ( 5 ) Atitoxidation product o f ( 4 ) ( 6 ) Ursodeoxycholic acid: sodium salt i n water ( 7 ) Ursodeoxycholic acid (free acid) in DMSO ( X ) Deouycholic acid: sodium salt in water ( 9 ) Deoxycholic acid (free acid) in DMSO ( 10) Lithocholic acid: sodium salt in water ( I I ) Lithocholic acid + nitrofurantoin ( 1 2 ) Ursodeoxycholic acid + nitrofurantoin ( 13) ('henodcoxycholic acid nitrofurantoin ( 14) Nitrofurantoin


0.00I 0.002 0.00I6 0.00 14


Negative 0.0026

N cga t Ivc 0 001 I Negative 7 40 7.30 6.0 5.0

activity ratio is normalized to a value in the absence of test compound, which is known as the induction factor. When the induction factor is plotted against concentration a linear region occurs. The slope of this region is called the SOS-inducing potency (SOSIP) and is a quantitative expression of the capacity to induce the .s/i'A response. These preliminary results (Table 1 ) reveal clTects not observed in the Ames test. The sodium salts of the parent bile acids show no genotoxic effects. The free acids in dimethyl sulphoxide (DMSO). however. show weak gcnotoxic effects, as do compounds ( I 5 ) . Co-mutagenic elrects are also seen with the parent bile acids when mixed with the known mutagen nitrofurantoin. This can be seen by an increase in the SOSIP valucs, compared with nitrofurantoin alone. We :ire grateful to Ilr. M I { o f n u n g for supplying the h;ictcri;il strain P o 3 7 and to the S . E R C.. for linnncinl support ol'ihi\ work.

Arniatrong. B. & I l o l l . R . (1975) / ) I / . J . C'crriwr 15. 617 631 Hill. M. J.. I h s c r . B. S . Aries. V.. ('rowthcr. J . S..Iiawksworth.

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