• Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling.

      Arlt, Volker M.; Poirier, Miriam C.; Sykes, Sarah E.; John, Kaarthik; Moserova, Michaela; Stiborova, Marie; Wolf, C. Roland; Henderson, Colin J.; Phillips, David H. (2012-09-03)
      Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por.
    • On the origins and development of the (32)P-postlabelling assay for carcinogen-DNA adducts.

      Phillips, David H. (2012-11-23)
      The (32)P-postlabelling method for the analysis of carcinogen-DNA adducts originated 30years ago from Baylor College of Medicine in Houston and was the work of a team comprised of Kurt and Erica Randerath, Ramesh Gupta and Vijay Reddy. With subsequent modifications and developments, it has become a highly sensitive and versatile method for the detection of DNA adducts that has been applied in a wide range of human, animal and in vitro studies. These include monitoring human exposure to environmental and occupational carcinogens, investigating genotoxicity of chemicals, elucidating pathways of metabolic activation of carcinogens, mechanistic studies of DNA repair, analysing the genotoxicity of complex mixtures and in ecotoxicology studies. Its use has been instrumental in providing new clues to the aetiology of some cancers and in identifying a new human carcinogen.
    • ³²P-HPLC analysis of N1-(2-carboxy-2-hydroxyethyl)deoxyadenosine: a DNA adduct of the acrylamide-derived epoxide glycidamide.

      Kotova, Natalia; Juren, Tina; Myohanen, Kirsi; Cornelius, Michael; Abramsson-Zetterberg, Lilianne; Backman, Josefin; Menzel, Ulrike; Rydberg, Per; Kronberg, Leif; Vahakangas, Kirsi; et al. (2011-11-10)
      Acrylamide (AA) is produced in many types of food products cooked or processed at high temperature. AA is metabolized to the epoxide glycidamide (GA), which can bind to deoxyguanosine and deoxyadenosine in DNA. The GA-derived N7-guanine and N3-adenine adducts are the only products which so far have been analysed in vivo. Because of previous excellent experience from analysis of adducts to N1-adenine, the aim of our study was to investigate if the N1-adenine adduct of GA could be used as a biomarker of AA exposure. A ³²P-postlabelling method was developed and tested (a) on DNA modified in vitro with GA, (b) on cells treated with GA and (c) on liver DNA from mice treated with AA. The N1-adenine adduct of GA (analysed after conversion to N⁶-GA-deoxyadenosine-5'-monophosphate) was easily detected in DNA reacted with GA and in DNA from cells exposed to GA, but not in DNA from mice treated with AA. The reason for this is currently not clearly understood, but some of the possible contributing factors are discussed. The application of the method in other experimental conditions should be further pursued in order to solve this matter.
    • Quantification of ultraviolet radiation-induced DNA damage in the urine of Swedish adults and children following exposure to sunlight.

      Liljendahl, Tove Sandberg; Kotova, Natalia; Segerback, Dan (2012-11)
      DNA damage following exposure to ultraviolet radiation (UVR) is important in skin cancer development. The predominant photoproduct, cyclobutane thymine dimer (T=T), is repaired and excreted in the urine, where it provides a biomarker of exposure.
    • Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: studies with the hepatic NADPH:cytochrome P450 reductase null mouse.

      Stiborova, Marie; Arlt, Volker M.; Henderson, Colin J.; Wolf, C. Roland; Kotrbova, Vera; Moserova, Michaela; Hudecek, Jiri; Phillips, David H.; Frei, Eva (2008-02-01)
      Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by (32)P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study.