Correlation between DNA or protein adducts and benzo[a]pyrene diol epoxide Itriglyceride adduct detected in vitro and in vivo
Seung Jun Kwack and
Byung Mu Lee1
Division of Toxicology, School of Pharmacy, SungKyunKwan University, Chunchun-Dong, Changan-Ku, Kyunggi-Do, Suwon 440-746, Korea
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Abstract
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In this study, we demonstrated the in vitro and in vivo formation of carcinogenlipid adduct and its correlation with DNA or protein adducts. The lipids from serum or hepatocyte membranes of SpragueDawley rats, human serum and standard major lipids were in vitro reacted with benzo[a]pyrene (B[a]P) and B[a]P metabolites. 7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I), an ultimate carcinogenic form of B[a]P, was covalently bound to triglyceride (TG). BPDE-ITG adducts isolated by thin-layer chromatography (TLC) were further detected by high-performance liquid chromatography. TGs, including triolein, tripalmitin and tristearin, showed positive reactions with BPDE-I. However, cholesterol, phospholipids (phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidyl-inositol and sphingomyelin) and non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid and stearic acid) did not react with BPDE-I. In addition, other B[a]P metabolites (B[a]P-phenols and -diols) did not react with TG. TG appeared to be the most reactive lipid yet studied with respect to its ability to form an adduct with BPDE-I. There was a clear-cut dose-related formation of [1,3-3H]BPDE-Ilipid adducts in vitro between TG and [1,3-3H]BPDE-I. In an animal study, BPDE-ITG was also formed in the serum of rats orally treated with B[a]P (25 mg/rat). Also, obvious correlations between [3H]B[a]P related-biomolecule adducts (DNA or protein) or lipid damage and the BPDE-ITG adducts were obtained in various tissues of mice i.p. treated with [3H]B[a]P. These data suggest that TG can form an adduct with BPDE-I, as do other macromolecules (DNA, RNA and protein). Therefore, a carcinogenlipid adduct would be a useful biomarker for chemical carcinogenesis research and cancer risk assessment.
Abbreviations: B[a]P, benzo[a]pyrene; BPDE-I, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (benzo[a]pyrene diol epoxide-I); HPLC, high-performance liquid chromatography; TG, triglyceride; THF, tetrahydrofuran; TLC, thin-layer chromatography.
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Introduction
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Exposure to carcinogens can cause damage to various biomolecules such as DNA, RNA and protein in the body, and such damage has been used as a biomarker (1). Biomarkers are defined as cellular, biochemical or molecular alterations in biological specimens of fluids, cells or tissues, and have been developed and applied to cancer research and risk assessment (2). In chemical carcinogenesis, the formation of a carcinogenDNA adduct is a critical step and is therefore considered an important biomarker during the initiation stage (3). CarcinogenDNA adducts have been measured in human tissues to predict exposure levels of carcinogens as well as to investigate the cause of carcinogenesis (4). However, the analysis of DNA adducts in human tissues has limitations in terms of sampling, sensitivity and specificity depending on the analytical methods (reviewed in refs 5 and 6).
Lipids are good sources of energy in the body and are composed of cellular membrane and tissue. There is some evidence that lipids could be involved in carcinogenesis. About 50% of all cancer patients show a syndrome of cachexia, characterized by the loss of adipose tissue and skeletal muscle mass (reviewed in ref. 7). Lipids are easily peroxidized to lipid peroxides by free radicals (8), and malondialdehyde is used to estimate lipid damage during lipid peroxidation. Lipid peroxidation products are also bound to DNA in the human liver and leukocytes (9,10). In recent studies, changes in the fatty acid composition of plasma lipids were characterized in patients with malignancy by nuclear magnetic resonance and gas-liquid chromatography (11,12), and lipid analysis was also performed in smooth muscle tumors (13). In breast cancer patients, serum triglyceride (TG), total cholesterol, high-density lipoprotein, low-density lipoprotein and very-low density lipoprotein levels were quantified in order to evaluate the changes in serum lipids and lipoproteins in the early and advanced stages of the disease. These studies were undertaken to assess the lipid changes due to the presence of malignancy and to investigate the usefulness of lipid changes for the diagnosis and prognosis of malignancy or metastasis (14,15). Pre-labelingchase experiments with [14C]arachidonic acid in human gastric cancer cells showed that arachidonic acid was preferentially incorporated into the TGs followed by a transfer to phospholipids (16). If lipids are damaged by exposure to carcinogens, their biosynthesis and metabolism would be influenced in the body. This leads to the question of whether lipid damage can be used as a biomarker or play any role in the process of chemical carcinogenesis.
For the above stated reasons, this study was undertaken to investigate whether benzo[a]pyrene diol epoxide (BPDE) can directly damage lipids to form BPDElipid adducts in vitro and in vivo. The correlation between DNA or protein adducts and BPDElipid adducts was also investigated.
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Materials and methods
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Chemicals
TG, triolein, tripalmitin, tristearin, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, sphingomyelin, cholesterol, palmitic acid, oleic acid, linoleic acid, stearic acid and benzo[a]pyrene were purchased from Sigma Chemical Co. (St Louis, MO). The TG assay kit was purchased from Sigma Diagnostics Co. (±)-r-7,t-8-Dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and other B[a]P metabolites (B[a]P-phenols and B[a]P-diols) were purchased from the National Cancer Institute (NCI), Chemical Carcinogen Reference Standard Repository at the Midwest Research Institute (Kansas, MO). [1,3-3H]BPDE-I (439 mCi/mmol) and [3H]B[a]P (66 Ci/mmol) were obtained from the NCI Radiochemical Carcinogen Reference Standard Repository at Chemsyn Science Laboratories (Lenexa, Kansas, MO).
Preparation of total lipid from animals and humans
The total lipid was prepared from hepatocytes or sera of SpragueDawley rats, and human sera. After a male rat (250 ± 20 g) was anesthetized, the abdomen was cut open and perfusion was initiated through the portal vein. Ca2+-free buffer and collagenase buffer were successively pumped from a reservoir through the portal vein at 37°C (17). The released hepatocytes were cultured in a CO2 incubator for 24 h and sound hepatocytes were separated. Whole blood (10 ml) was taken from the animals or human volunteers (mean age: 24 ± 2, n = 10) and the serum was separated promptly after centrifugation at 3000 r.p.m. for 20 min. Folch solution [chloroform:methanol, 2:1 (v/v)] was added to hepatocytes or serum and agitated for 3 h. After centrifugation at 3000 r.p.m. for 10 min, the lower layer was separated and the concentration of total lipid was determined by UV absorption spectroscopy at 590 nm as described in the dichromate oxidation method (18). The total lipid was dissolved in tetrahydrofuran (THF) at a concentration of 1 mg/ml. The male rats (250 ± 20 g) were orally treated with B[a]P (25 mg/rat) in 2 ml of corn oil to investigate B[a]Plipid adducts in vivo. After 6 h, the rats were anesthetized and 10 ml of whole blood was taken from the abdominal aorta. Other procedures were as above.
In vitro detection of lipid adducts
Standard lipids (cholesterol, TG, triolein, tripalmitin, tristearin, palmitic acid, oleic acid, linoleic acid, stearic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and sphingomyelin; 50 µg each in 50 µl THF or chloroform) or total lipids from animals and humans were incubated with B[a]P or B[a]P metabolites (50 µl, 1 mg/ml THF or chloroform) for 2 h at 37°C. After incubation, each sample was applied as a 1 cm streak to thin-layer chromatography (TLC) plates (silica gel 60; Merck, Darmstadt, Germany) and the solvent front was allowed to migrate to a predetermined boundary mark. The cholesterol and TG were separated using a THF/hexane two component system: THF/hexane/formic acid (20:80:2 by vol.) to 18 cm above the origin and THF/hexane/formic acid (100:20:2 by vol.) to 10 cm (19,20). Total lipids, phospholipids and free fatty acids were separated using a three-development system: diethylether/hexane/ammonia (50:50:0.25 by vol.) to 18 cm above the origin, chloroform/methanol/water/ammonia (70:30:3:2 by vol.) to 10 cm and ethylacetate/acetone/water/glacial acetic acid (40:40:2:1 by vol.) to 16 cm (21). After TLC the plates were dried in a desiccator, and the spots were located under UV light (254 and 365 nm) or observed after spraying with 10% sulfuric acid. The Rf values from the reaction of TG, tripalmitin, triolein and tristearin with BPDE-I were all 0.88.
Analysis of lipid adducts and modification levels by HPLC
Each standard lipid (50 µg) was mixed with [1,3-3H]BPDE-I (3.5 nmol) in THF for 2 h at 37°C and was applied to a TLC plate. Solvent systems were as described above. All spots were scraped off into a screw-cap scintillation vial and the radioactivity was measured using a liquid scintillation counter (LSC, Pharmacia-LKB 1209 Rackbeta, Uppsala, Sweden). The modification level of the [1,3-3H]BPDE-ITG adduct was determined on the basis of the molar ratio of [1,3-3H]BPDE-I to TG. For HPLC analysis, the reaction mixture was centrifuged and the supernatant was filtered through a 0.45 µm membrane prior to injection into an Econosil silica column (250x10 mm inner diameter; 10 µm particle size; Alltech, Deerfield, Illinois) attached to an HPLC system (Gilson 805; Gambetta, France) using a THF/hexane solvent system (22,23).
In vivo doseresponse relationships for B[a]P adducts with DNA, protein, lipid and TG in different mouse tissues
ICR mice (32 ± 2 g; three animals/dose) were injected i.p. with 0.33, 1 or 3 mg [3H]B[a]P (66 Ci/mmol) in 0.5 ml corn oil. After 24 h, the mice were killed and the liver, heart, lung, stomach, kidney, spleen and brain were collected. The organs were washed three times with 0.9% saline and, after homogenization, were incubated (37°C, 1 h) in 4 vol of 10% SDS buffer solution. The total lipid and protein were separated using a Folch solution, and DNA was isolated from the tissues using a standard phenol, chloroform/isoamyl alcohol extraction, and RNase treatment (24). The concentration of TG was measured in the total lipid using the TG assay kit. All samples were counted on an LSC at a concentration of 1 mg/ml.
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Results
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To investigate the formation of carcinogenlipid adducts, total lipids were prepared from rat serum or hepatocyte membrane and human serum, and were reacted with B[a]P and BPDE-I. TLC showed that BPDE-I, but not B[a]P, covalently bound to a certain lipid regardless of the lipid source (Figure 1
). On the TLC plates, a new spot (spot F) was formed with a greenish fluorescence, whereas BPDE-I fluoresces blue under the UV light (365 nm). To further investigate BPDE-Ilipid damage, standard lipids (cholesterol, TGs, phospholipids and free fatty acids) were reacted with BPDE-I and the Rf value of each spot produced by TLC was estimated. Only TG, tripalmitin, triolein and tristearin reacted with BPDE-I and produced a BPDE-ITG adduct spot on the TLC plates, similar to the BPDETG adduct spot observed in Figure 1
.

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Fig. 1. TLC analysis of total lipid treated in vitro with B[a]P and BPDE-I. (A) B[a]P; (B) BPDE-I; (C) lipid of rat serum; (D) B[a]P mixed with (C); (E) BPDE-I mixed with (C); (F) BPDE-Ilipid adducts spot; (G) TG.
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The formation of a BPDE-ITG adduct was reconfirmed using [1,3-3H]BPDE-I. A two-dimensional TLC was developed for a reaction mixture of TG (50 µg) and [1,3-3H]BPDE-I (1.5x106 c.p.m.). Four different spots were detected and the c.p.m. values of each spot were measured by LSC (Figure 2
). Spots A and B were BPDE-I (two separate regions in this solvent system), spot C was BPDE-ITG adduct, and spot D was TG, which was confirmed by separate TLC application of BPDE-I and TG. The radioactivities of the spots were as follows: A, 423232; B, 44862; C, 253215 and D, 151 c.p.m.

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Fig. 2. Two-dimensional TLC analysis of TG was reacted with [1,3-3H]BPDE-I. (A and B) [1,3-3H]BPDE-I isomers; (C) [1,3-3H]BPDE-ITG adducts; (D) TG. Samples were two-dimensionally (arrows 1 and 2) developed from the origin.
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In a doseresponse experiment, [1,3-3H]BPDE-ITG was increasingly formed in vitro as a function of [1,3-3H]BPDE-I dose (Figure 3
). In a timeresponse experiment, the [1,3-3H]BPDE-ITG adduct reached a peak level 24 h after incubation at 37°C and remained at this level for 48 h (data not shown). The formation of the [1,3-3H]BPDE-ITG adduct was further detected by HPLC analysis and chromatograms of the separations are shown in Figure 4
.

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Fig. 3. Dose-related formation of [1,3-3H]BPDE-ITG in TG (50 µg) treated with [1,3-3H]BPDE-I. The sensitivity of TLC to detect BPDE-ITG was 20 pmol BPDE-I/µg total lipid.
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Fig. 4. HPLC analysis of BPDE-ITG adduct in TG treated with BPDE-I. (A) TG; (B) BPDE-I; (C) BPDE-ITG adduct. Samples were eluted at a flow rate of 3 ml/min and detected at 280 nm with a sensitivity of 1.0 mA. Solvent A [hexane, 1% methanol, 0.5% tetraethylammonium (TEA)] and B (THF, 1% methanol, 0.5% TEA) were used as a gradient mobile phase system: 40% B was used as the eluent for the first 25 min, and then increased to 100% B during the next 15 min, and remained at 100% B for 30 min.
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In animal experiments, a B[a]P-metabolitelipid adduct was formed in the serum of rats orally treated with B[a]P (25 mg/rat). The fluorescent hue under UV light and the Rf value of the B[a]P-metabolitelipid adduct so formed, were identical to those of [1,3-3H]BPDE-ITG formed in vitro (spot F in Figure 1
). The correlation coefficients of the B[a]PDNA/B[a]PTG adducts or the B[a]Pprotein/B[a]PTG adducts were 0.99 for all tissues (Table I
).
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Table I. Doseresponse relationships for [3H]B[a]P adducts to DNA, protein, lipid and TG in various tissues of mice i.p. treated with [3H]B[a]P
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Discussion
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Biomolecular damage is widely believed to be a critical event during mutagenesis and carcinogenesis. Carcinogenmacromolecule adducts (DNA, RNA or protein adducts) have been largely studied with known chemical carcinogens (24). However, the analysis of DNA or protein adducts in human tissues still has limitations in terms of sampling, sensitivity and specificity. DNA or protein adducts have been measured in human tissues for the etiological investigation of carcinogen exposure and qualitative risk assessment. Lipids are abundant in human tissues and may be susceptible to the exposure of carcinogens. If carcinogen-derived lipid damage occurs, it could be useful as an alternative to DNA or protein adducts.
In this study, BPDElipid adduct formation was detected in vitro and in vivo by the exposure of BPDE-I or B[a]P. The formation of the BPDElipid adduct in vitro indicated that the TGs, including tripalmitin, triolein and tristearin, are likely target lipids for BPDE-I. TGs (also called fats, triglycerols and depot lipids) constitute 90% of dietary lipids and are the major form of metabolic energy storage in humans (40150 mg/100 ml blood in adults). These non-polar, water-insoluble substances consist of glycerol triesters of fatty acids such as palmitic, oleic and stearic acids (25).
In the TGs of human lipids, palmitate tends to be concentrated at position 1 and oleate at position 2 of the base glycerol structure (26). Considering the structure of the TG species, a possible explanation for the formation of the BPDE-ITG adduct might be the covalent binding of position 10 of BPDE-I to the ester bonds of TG between the glycerol portion and free fatty acids of TG, though this must be investigated further. One important role of TG is transporting free fatty acids, either to various tissues or during the biosynthesis of several lipids in the body (27,28). Therefore, the adduct formation between BPDE-I and TG may be of biological significance and it may also serve as an index of any internal damage caused by carcinogens. In vivo correlations between either the biomolecular adducts (DNA, protein) or damage (lipid, in this case as B[a]P, B[a]P metabolites, and BPDE-ITG adducts are expected to be incorporated into the lipid) and TG adduct formation are obvious such that the lipid damage or TG adducts could serve as a surrogate biomarker for DNA or protein adducts.
These preliminary data suggest that the formation of the BPDE-ITG adduct may be an important piece of evidence that lipids (especially esterified forms) can be directly damaged by reactive carcinogens. The carcinogenlipid adduct or lipid damage may be useful biomarkers for carcinogen exposure, although carcinogenlipid adducts are limited to a TG adduct in this study. It is of interest whether the formation of a carcinogenlipid adduct plays a role in the process of chemical carcinogenesis. In the future, further investigation is needed on the chemical structure of BPDE-ITG, its biological role and any correlation with carcinogenDNA or carcinogenprotein adduct formation in human tissues. In addition, the development of better technology to detect TG adducts could facilitate their use for cancer research as well as risk assessment.
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Acknowledgments
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This work was supported by the Research Fund for Environmental Technology Development, Korean Environmental Protection Agency.
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Notes
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1 To whom correspondence should be addressed Email: bmlee{at}yurim.skku.ac.kr 
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References
|
---|
-
Wogan,G.N. (1989) Markers of exposure to carcinogens. Environ. Health Perspect., 81, 917.[ISI][Medline]
-
Bartsch,H., Rojas,M., Alexandrov,K. and Risch,A. (1998) Impact of adduct determination on the assessment of cancer susceptibility. Cancer Res., 154, 8696.
-
Dipple,A. (1995) DNA adducts of chemical carcinogens. Carcinogenesis, 16, 437441.[ISI][Medline]
-
Swenberg,J.A., Richardson,F.C., Boucheron,J.A. and Dyroff,M.C. (1985) Relationship between DNA adduct formation and carcinogenesis. Environ. Health Perspect., 62, 177183.[ISI][Medline]
-
Santella,R.M. (1999) Immunological methods for detection of carcinogenDNA damage in humans. Cancer Epidemiol. Biomarkers Prev., 8, 733739.[Free Full Text]
-
Kriek,E., Rojas,M., Alexandrov,K. and Bartsch,H. (1998) Polycyclic aromatic hydrocarbonDNA adducts in humans: relevance as biomarkers for exposure and cancer risk. Mutat Res., 400, 215231.[ISI][Medline]
-
Tisdale,M.J. (1997) Biology of cachexia. J. Natl Cancer Inst., 89, 17631773.[Abstract/Free Full Text]
-
Minotti,G., Mancuso,C., Frustaci,A., Mordente,A., Santini,S.A., Calafiore,A.M., Liberi,G. and Gentiloni,N. (1996) Paradoxical inhibition of cardiac lipid peroxidation in cancer patients treated with doxorubicinPharmacologic and molecular reappraisal of anthracycline cardiotoxicity. J. Clin. Invest., 98, 650661.[Abstract/Free Full Text]
-
Chaudhary,A.K., Nokubo,M., Reddy,G.R., Yeola,S.N., Morrow,J.D., Blair,I.A. and Marnett,L.J. (1994) Detection of endogenous malondialdehydedeoxyguanosine adducts in human liver. Science, 265, 15801582.[ISI][Medline]
-
Leuratti,C., Singh,R., Lagneau,C., Farmer,P.B., Plastaras,J.P., Marnett,L.J. and Shuker,D.E. (1998) Determination of malondialdehyde-induced DNA damage in human tissues using an immunoslot blot assay. Carcinogenesis, 19, 19191924.[Abstract]
-
Engan,T. (1995) Characterization of plasma lipids in patients with malignant disease by 13C-nuclear magnetic resonance spectroscopy and gas liquid chromatography. Blood, 85, 13231330.[Abstract/Free Full Text]
-
Kriat,M., Vion-Dury,J., Confort-Gouny,S., Favre,R., Viout,P., Sciaky,M., Sari,H. and Cozzone,P.J. (1993) Analysis of plasma lipids by NMR spectroscopy: Application to modifications induced by malignant tumors. J. Lipid Res., 34, 10091019.[Abstract]
-
Sivaraja,M., Turner,C., Souza,K. and Singer,S. (1994) Ex vivo two-dimensional proton nuclear magnetic resonance spectroscopy of smooth muscle tumors: advantages of total correlated spectroscopy over homonuclear J-correlated spectoscopy. Cancer Res., 54, 60376040.[Abstract]
-
Kökoglu,E., Karaarslan,I., Karaarslan,H.M. and Baloglu,H. (1994) Alteration of serum lipids and lipoproteins in breast cancer. Cancer Lett., 82, 175178.[ISI][Medline]
-
Saarto,T., Blomqvist,C., Ehnholm,C., Taskinen,M.R. and Elomaa,I. (1996) Antiatherogenic effects of adjuvant antiestrogens: a randomized trial comparing the effects of tamoxifen and toremifene on plasma lipid levels in postmenopausal women with node-positive breast cancer. J. Clin. Oncol., 14, 429433.[Abstract]
-
Najid,A., Es-Saady,D. and Denizot,Y. (1994) The decreased transfer of arachidonic acid from triglycerides to phospholipids during proliferation of a human gastric tumor (HGT) cell line is not linked to modification of membrane fluidity. Cancer Lett., 79, 189192.[ISI][Medline]
-
Seglen,P.O. (1976) Preparation of isolated rat liver cells. In Prescott,D.M. (ed.) Methods in Cell Biology. Academic Press, New York, pp. 3078.
-
Folch,J., Lees,M. and Stanley,G.H.S. (1957) A simple methods for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226, 497509.[Free Full Text]
-
White,T., Bursten,S., Federighi,D., Lewis,R.A. and Nudelman,E. (1998) High-resolution separation and quantification of neutral lipid and phospholipid species in mammalian cells and sera by multi-one-dimensional thin-layer chromatography. Anal. Biochem., 258, 109117.[ISI][Medline]
-
Entezami,A.A., Venables,B.J. and Daugherty,K.E. (1987) Analysis of lipids by one-dimensional thin-layer chromatography. J. Chromatogr., 387, 323331.[Medline]
-
Irvine,R.F., Letcher,A.J., Meade,C.J. and Dawson,R.M. (1984) One-dimensional thin-layer chromatographic separation of the lipids involved in arachidonic acid metabolism. J. Pharmacol. Methods, 12, 171182.[ISI][Medline]
-
Sewell,P.A. (1992) High-performance liquid chromatography. In Hamilton,R.J. and Hamilton,S. (eds) Lipid Analysis: A Practical Approach. Oxford University Press, New York, pp. 153203.
-
Clark,A.P., Longfellow,D.G. and Seifried,H.E. (1993) Analytical and spectra data. In Clark,A.P. (ed.) Handbook of Analytical and Spectral Data for Polycyclic Aromatic Hydrocarbons, Vol. I; Benzo(a)pyrene and its Metabolites. Midwest Research Institute, Kansas, MO, pp. 4143.
-
Pelling,J.C., Slaga,T.J. and DiGiovanni,J. (1984) Formation and persistence of DNA, RNA, protein adducts in mouse skin exposed to pure optical enantiomers of 7ß,8
-dihydroxy-9
,10
-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene in vivo. Cancer Res., 44, 10811086.[Abstract]
-
Christie,W.W. and Moore,J.H. (1970) Comparison of the structures of triglycerides from various pig tissues. Biochim. Biophys. Acta, 210, 4656.[ISI][Medline]
-
Brockerhoff,H., Hoyle,R.J. and Wolmark,N. (1966) Positional distribution of fatty acids in triglycerides of animal depot fats. Biochim. Biophys. Acta, 116, 6772.[ISI][Medline]
-
Voet,D. and Voet,J.G. (1990) Lipid metabolism. In Voet,D. and Voet,J.G. (eds) Biochemistry. Wiley, New York, pp. 618677.
-
Hole,J.W.Jr (1993) Nutrition and metabolism. In Wheatley,C.H. (ed.) Human Anatomy & Physiology. WCB, Iowa, pp. 548580.
Received August 24, 1999;
revised December 2, 1999;
accepted December 20, 1999.