Chapter 28 4
Detection of O -Alkylthymine in Human Liver DNA Molecular Epidemiological Study on Human Cancer Downloaded via NORTHWESTERN UNIV on July 11, 2018 at 15:58:10 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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Nam-ho Huh , Chieko Moriyama , Masahiko S. Satoh , Junji Shiga , and Toshio Kuroki 1
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Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan Department of Pathology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan
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Quantitation of premutagenic DNA structural modifications is among the most reliable markers for the assessment of possible human exposure to environmental carcinogens. Considering the ubiquitous distribution of alkylating N-nitroso compounds and their capability to induce malignant tumors in a wide variety of animal tissues, we quantitated O -ethylthymine in human liver DNA. Among 33 cases analysed, O -ethylthymine was detected in 30 cases and the mean O -ethylthymine level of 19 cancer cases was significantly higher than that of 11 non-cancerous cases. Further, when we screened 5 human liver DNA samples for some other premutagenic O-alkylated DNA adducts, Ο -methylthymine was detected in 3 cases, but none of the cases showed any detectable Ο -methyl- or Ο -ethylguanine levels. These results indicate that humans are actually exposed to alkylating N-nitroso compounds and Ο -alkylthymine may be a suitable marker for monitoring the exposure because of its stable nature in mammalian cells. For more extensive studies in this direction, we have established a highly sensitive and specific method to detect O -ethylthymine by a combination of pre-fraction by HPLC, 32P -postlabelling, and immunoprecipitation with monoclonal antibody (PREPI), thus increasing the sensitivity about 30 fold compared to our previous method without sacrifying its extremely high specificity. 4
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Chemical carcinogenesis is a complex multistep process involved in the induction of a large proportion of human cancers. Among the most important factors in this process are structural modifications of DNA by chemical carcinogens and their subsequent repair, the Abbreviations; 0 -EtG, 0 -ethylguanine; 0 -EtT, Ο -ethylthymine; Ο -EtdThd, Ο -ethyl-2 ' -dœxythymidine ; Ο -Et-3 '-ΤΜΡ, Ο - e t h y l ^ d e o x y t i i y i a i à i J i e ^ Ο -MetG, Ο ^ethylguanine; ΟMetT, Ο ^etliylthymidine; Τ, thymidine; PREPI, A detection method by pre fractionation, postlabeling and immunoprecipitation. 4
0097-6156/91/0451-0308$06.00/0 © 1991 American Chemical Society Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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Detection of 0*~Alkylthymine in Human Liver DNA
e f f i c i e n c y o f which d i f f e r s w i d e l y depending on t h e t y p e o f DNA a d d u c t s , c e l l s , t i s s u e s , and a n i m a l s p e c i e s ( 1 - 3 ) . In t h i s r e g a r d , a l k y l a t i n g N - n i t r o s o compounds a r e most w e l l s t u d i e d , and a number^ of specific DNA modifications including 0 -alkylguanine and 0 -alkylthymine are identified as premutagenic and/or pretransf o r m a t i o n a l l e s i o n s (2, 4-10). In a t r a n s p l a c e n t a l c a r c i n o g e n e s i s with a pulse treatment of N-ethyl-N-nitrosourea i n r a t s , Goth and Rajewsky showed t h a t p e r s i s t a n c e o f 0 - e t h y l g u a n i n e (O -EtG) in c e l l u l a r DNA may be r e s p o n s i b l e f o r t h e p a r t i c u l a r s u s c e p t i b i l i t y o f b r a i n c e l l s t o m a l i g n a n t c o n v e r s i o n (2^. T h i s n o t i o n was f u r t h e r c o n f i r m e d by an i n v i t r o s t u d y , where t r a n s f o r m a t i o n f r e q u e n c y was compared i n a ^ e t o f c l o n a l f i b r o b l a s t s w i t h different repair c a p a c i t i e s f o r 0 - E t G (Thomale e t a l . , m a n u s c r i p t i n p r e p a r a t i o n ) . The r e p a i r - p r o f i c i e n t v a r i a n t s were f a r more r e s i s t a n t t o m a l i g n a n t conversion by N - e t h y l n i t r o s o u r e a , but n o t by ben ζ ( a ) p y r e n e - 7 , 8 diol-9,10-epoxide, than the r e p a i r - d e f i c i e n t c l o n e . On t h e o t h e r hand, c o n t i n u o u s feeding of r a t s with dLethylnitrosamine l e a d to accumulation of 0 -ethylthymine (0 - E t T ) i n target hepatocytes, while 0 -Et(^ content i n t h e DNA r e m a i n e d a t about 50 f o l d lower l e v e l t h a n 0 - E t T because o f r a p i d e n z y m a t i c e l i m i n a t i o n ( 10 ). D e t e c t i o n o f such c r i t i c a l m o d i f i c a t i o n s i n DNA d e r i v e d from human t i s s u e s may be a r e l i a b l e marker f o r m o n i t o r i n g human e x p o s u r e to environmental a l k y l a t i n g a g e n t s , and contribute to possible i d e n t i f i c a t i o n o f h i g h - r i s k group o r i n d i v i d u a l s who may develop cancer i n the f u t u r e . S i n c e humans a r e e x p o s e d c h r o n i c a l l y t o low c o n c e n t r a t i o n o f d i v e r s e e n v i r o n m e n t a l c a r c i n o g e n s , DNA a d d u c t s i n human t i s s u e s a r e e x p e c t e d t o be a complex m i x t u r e o f d i f f e r e n t modifications at extremely low levels. Highly sensitive and s p e c i f i c methods a r e , t h e r e f o r e , e s s e n t i a l f o r p r e c i s e q u a n t i t a t i o n o f DNA a d d u c t s i n human m a t e r i a l s . In t h i s paper, we d e s c r i b e a s t u d y on t h e d e t e c t i o n o f 0 - E t T i n human l i v e r DNA u s i n g HPLC f r a c t i o n a t i o n and r a t i o i m m u n o a s s a y (RIA), and t h e development o f a detection method for 0 -EtT with improved sensitivity and specificity. 6
Table
I.
Ο - E t T i n human l i v e r
DNA
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o - E t T / T (x 10*>> No. o f cases analysed
Cases I Liver
cancer
II Cancer i n other organs I I I Nonmalignancy a
Detection limit
Difference and I I I : p
