XENOBIOTICA,
1990, VOL. 20,
NO.
9, 901-907
A review of recent studies on the metabolism of exogenous and endogenous malondialdehyde H. H. DRAPER and M. HADLEY Department of Nutritional Sciences, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Xenobiotica Downloaded from informahealthcare.com by National University of Singapore on 06/17/14 For personal use only.
Received 17 October 1989; accepted 14 February 1990 1. The generation of malondialdehyde (MDA), a mutagenic product of the oxidative decomposition of highly unsaturated fatty acids in vivo, is increased by exposure to certain environmental oxidants and xenobiotics. 2. This increase is reflected in enhanced excretion of several MDA derivatives in the urine. The main urinary metabolites of MDA have been identified as N-&-(2propena1)lysine and its N-a-acetyl ester. 3. Two minor metabolites have been identified as enaminals formed by reactions with the phospholipid bases serine and ethanolarnine. A further MDA metabolite has been identified as a cyclized adduct with guanine. 4. These urinary compounds reflect the turnover of proteins, phospholipids and nucleic acids that have been modified by reactions with MDA in viuo. Monitoring of the urinary adduct with guanine may provide a practicable method of assessing the effect of xenobiotics and other factors on in vivo lipid peroxidation. 5 . The proportion of total MDA in the diet, blood, urine and solid tissues that exists in the free state appears to be negligible. 6. Chronic oral administration of the enol Na salt of MDA to animals produced no significant pathology except for dose-dependent lesions of hepatic nuclei. Nuclear abnormalities in cultured rat skin fibroblastswere seen at intracellular concentrations as low as ~ o - ~ M .
Introduction Interest in the metabolism of malondialdehyde (MDA) (figure 1)stems from its mutagenicity (Basu and Marnett 1983) and purported carcinogenicity (Shamberger et al. 1974). Exposure to exogenous MDA arises from its occurrence as a product of the decomposition of peroxidized polyunsaturated fatty acids (PUFA) in foods. Endogenous MDA is generated by the decomposition of analogous fatty acyl peroxides in the phospholipids of cell membranes and as a product of the cyclooxygenase reaction in prostaglandin synthesis. The endogenous production of MDA is increased by environmental factors that stimulate lipid peroxidation in viwo, such as exposure to ozone, nitrogen oxides, hyperoxia and certain xenobiotics, as well as by changes to the internal milieu such as the accumulation of highly unsaturated acids, vitamin E or selenium deficiency, and tissue injury that results in the release of metal catalysts of lipid peroxidation. The metabolism of exogenous MDA differs significantly from that of endogenous MDA (PichC et al. 1988a), and there are undoubtedly differences in the metabolism of endogenous MDA depending upon the site of its generation.
Toxicity of exogenous MDA in cultured cells Controversy has surrounded the mutagenicity and carcinogenicity of MDA because of the occurrence of mutagenic side-products, such as b-methoxyacrolein, in 0049-8254/90 $300 0 1990 Taylor &. Francis Ltd.
H . H . Draper and M . Hadley
902 0
\\
F-cHz-c H
//o
\ H
\H
Malondialdehyde
N-(2-Propena1)ethanolamine
N\"z CH- CH2- CHz- CH2-CHz-NH /
-CH=CH-C
o=c
2 \ H
\OH
Xenobiotica Downloaded from informahealthcare.com by National University of Singapore on 06/17/14 For personal use only.
CHzOH
o=c
\CH-NH-CH=CH/
c/p \ H
\OH
N-r-(8-Propena1)lysine
c\H3 /c=o
HN
'+cHz-~~z-~~z-
o=c
P
CHZOH-CHz-NH-CH=CH-C
cHZ--~H-- CH=CH-
/
N-(2-Propenal)serine
P
C,
\
H
Guanine-rnalondialdehyde adduct
\OH I
N-a-Acetyl-c-(2-propena1)lysine
Figure 1 . Urinary metabolites of malondialdehyde. N-e-(2-Propenal)lysine and its N-a-acetyl derivative are the main MDA derivatives excreted in urine. They are mainly, but not entirely, of dietary origin and their proportions vary markedly depending on the extent of acetylation. The serine and ethanolamine adducts reflect reactions of MDA with their corresponding phospholipids, and the guanine adduct with nucleic acids. These three metabolites appear to be exclusively of endogenous origin. Minor metabolites that yield MDA on acid hydrolysis remain unidentified.
crude MDA test material prepared by hydrolysis of its stable precursor tetramethoxypropane (Marnett and Tuttle 1980). Application of such material plus a promotor to the shaved backs of mice produced a high incidence of skin and liver tumours (Shamberger et al. 1974). Whether pure MDA or its enol sodium salt elicits a similar response is unknown. However, MDA has been shown to be an authentic, though not highly potent, mutagen in Salmonella typhimwium (Basu and Marnett 1983). Mutagenicity also has been reported in Escherichia coli (Yonei and Furui 1981) and a mammalian lymphoma cell line (Yau 1979). The relevance of MDA toxicity tests conducted on whole cells or on animals for MDA generated in vivo is questionable. Although a relatively small dose of MDA (2 pg/g body weight) administered orally to rats results in the appearance of traces of free MDA in the urine (McGirr et al. 1985), little or no free MDA is detectable in normal human plasma, urine or solid tissues. A negligible fraction of MDA in foods is absorbed in the free form (PichC et al. 1988a) and hence it is unlikely that the diet is a significant source of free MDA in the tissues. The relative benignancy of MDA administered orally therefore may have little relevance to the toxicity of MDA formed, for example, in the nuclear membrane in proximity to the genome, by stimulation of lipid peroxidation by environmental factors. The effects of adding MDA to a culture medium for rat skin fibroblasts are listed in table 1 (Bird and Draper 1980, Bird et al. 1982a). Studies on [1,3-I4C]MDA metabolism indicated that, as a result of binding to proteins in the medium, MDA uptake was only 4%. The nuclear effects seen at concentrations of lo-' to M in the medium therefore may have occurred at intracellular concentrations as low as
Metabolism of malondialdehyde
903
Table 1 . Effects of adding MDA as the enol Na salt to the medium of rat skin fibroblasts grown in culture’. Treatment ~ O - ’ M , 120h
1 0 - 4 ~12h ,
Xenobiotica Downloaded from informahealthcare.com by National University of Singapore on 06/17/14 For personal use only.
1 0 - 4 ~120h , to 1 0 - 6 ~3h , to 1 0 - 6 ~120h ,
Effects Altered morphology, cytoplasmic vacuolization, karyorrhexis, micro- and multinucleation, decreased mitotic index, decreased DNA, RNA and protein synthesis Chromosomal fragments, achromatic lesions, chromatid breaks Mitotoc aberrations, micronucleation,decreased mitotic index, decreased DNA Increased DNA repair synthesis Small and irregular nuclei
’Bird and Draper 1980, Bird et al. 1982a. Studies on [1,2-’*C]MDA uptake showed that -95% of the radioactivity remained, bound to protein, in the medium.
~ O - ’ M . The material used in these cell culture studies, as well as in the animal studies cited below (Bird et al. 1982b, Siu et al. 1983), was purified by recrystallization three times from acetone and shown to be free of side-products (Marnett and Tuttle 1980) by chromatography on Sephadex LH-20.
Toxicity of oral M D A The acute oral LD,, of MDA for rats, administered as the enol Na salt (Na oxyacrolein), has been found to be 632pglg body weight (Crawford et al. 1965). Administration of MDA to mature Swiss mice at levels of 2,10, 50,250 or 500 pg/g body weight per day for 90 days resulted in no general histopathology (Siu et al. 1983). However, the liver nuclei exhibited dose-dependent irregularities including anisokaryosis, hyperchromicity and vesiculation, and at the highest level there was weight loss and atrophy of the exocrine cells of the pancreas with loss of zymogen granulation. Administration of MDA as the enol Na salt in the drinking water of Swiss mice at levels of 0.1,l and 10pg/g body weight per day for 12 months resulted in no increase in the incidence of lesions in 27 tissues examined, except for the liver (Bird et al. 1982 b). Hepatic abnormalities, observed at the two highest doses, included anisokaryosis, changes in cytoplastic volume with architectural derangements, necrosis, and neoplastic changes (nodular hyperplasia, hepatoma and haemangioma). There was no increase in specific neoplasms, but the incidence of total neoplasms and neoplastic lesions was dose-dependent (4%, 8% and 12%, respectively, vs. 1% in controls) (P
1990, VOL. 20,
NO.
9, 901-907
A review of recent studies on the metabolism of exogenous and endogenous malondialdehyde H. H. DRAPER and M. HADLEY Department of Nutritional Sciences, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Xenobiotica Downloaded from informahealthcare.com by National University of Singapore on 06/17/14 For personal use only.
Received 17 October 1989; accepted 14 February 1990 1. The generation of malondialdehyde (MDA), a mutagenic product of the oxidative decomposition of highly unsaturated fatty acids in vivo, is increased by exposure to certain environmental oxidants and xenobiotics. 2. This increase is reflected in enhanced excretion of several MDA derivatives in the urine. The main urinary metabolites of MDA have been identified as N-&-(2propena1)lysine and its N-a-acetyl ester. 3. Two minor metabolites have been identified as enaminals formed by reactions with the phospholipid bases serine and ethanolarnine. A further MDA metabolite has been identified as a cyclized adduct with guanine. 4. These urinary compounds reflect the turnover of proteins, phospholipids and nucleic acids that have been modified by reactions with MDA in viuo. Monitoring of the urinary adduct with guanine may provide a practicable method of assessing the effect of xenobiotics and other factors on in vivo lipid peroxidation. 5 . The proportion of total MDA in the diet, blood, urine and solid tissues that exists in the free state appears to be negligible. 6. Chronic oral administration of the enol Na salt of MDA to animals produced no significant pathology except for dose-dependent lesions of hepatic nuclei. Nuclear abnormalities in cultured rat skin fibroblastswere seen at intracellular concentrations as low as ~ o - ~ M .
Introduction Interest in the metabolism of malondialdehyde (MDA) (figure 1)stems from its mutagenicity (Basu and Marnett 1983) and purported carcinogenicity (Shamberger et al. 1974). Exposure to exogenous MDA arises from its occurrence as a product of the decomposition of peroxidized polyunsaturated fatty acids (PUFA) in foods. Endogenous MDA is generated by the decomposition of analogous fatty acyl peroxides in the phospholipids of cell membranes and as a product of the cyclooxygenase reaction in prostaglandin synthesis. The endogenous production of MDA is increased by environmental factors that stimulate lipid peroxidation in viwo, such as exposure to ozone, nitrogen oxides, hyperoxia and certain xenobiotics, as well as by changes to the internal milieu such as the accumulation of highly unsaturated acids, vitamin E or selenium deficiency, and tissue injury that results in the release of metal catalysts of lipid peroxidation. The metabolism of exogenous MDA differs significantly from that of endogenous MDA (PichC et al. 1988a), and there are undoubtedly differences in the metabolism of endogenous MDA depending upon the site of its generation.
Toxicity of exogenous MDA in cultured cells Controversy has surrounded the mutagenicity and carcinogenicity of MDA because of the occurrence of mutagenic side-products, such as b-methoxyacrolein, in 0049-8254/90 $300 0 1990 Taylor &. Francis Ltd.
H . H . Draper and M . Hadley
902 0
\\
F-cHz-c H
//o
\ H
\H
Malondialdehyde
N-(2-Propena1)ethanolamine
N\"z CH- CH2- CHz- CH2-CHz-NH /
-CH=CH-C
o=c
2 \ H
\OH
Xenobiotica Downloaded from informahealthcare.com by National University of Singapore on 06/17/14 For personal use only.
CHzOH
o=c
\CH-NH-CH=CH/
c/p \ H
\OH
N-r-(8-Propena1)lysine
c\H3 /c=o
HN
'+cHz-~~z-~~z-
o=c
P
CHZOH-CHz-NH-CH=CH-C
cHZ--~H-- CH=CH-
/
N-(2-Propenal)serine
P
C,
\
H
Guanine-rnalondialdehyde adduct
\OH I
N-a-Acetyl-c-(2-propena1)lysine
Figure 1 . Urinary metabolites of malondialdehyde. N-e-(2-Propenal)lysine and its N-a-acetyl derivative are the main MDA derivatives excreted in urine. They are mainly, but not entirely, of dietary origin and their proportions vary markedly depending on the extent of acetylation. The serine and ethanolamine adducts reflect reactions of MDA with their corresponding phospholipids, and the guanine adduct with nucleic acids. These three metabolites appear to be exclusively of endogenous origin. Minor metabolites that yield MDA on acid hydrolysis remain unidentified.
crude MDA test material prepared by hydrolysis of its stable precursor tetramethoxypropane (Marnett and Tuttle 1980). Application of such material plus a promotor to the shaved backs of mice produced a high incidence of skin and liver tumours (Shamberger et al. 1974). Whether pure MDA or its enol sodium salt elicits a similar response is unknown. However, MDA has been shown to be an authentic, though not highly potent, mutagen in Salmonella typhimwium (Basu and Marnett 1983). Mutagenicity also has been reported in Escherichia coli (Yonei and Furui 1981) and a mammalian lymphoma cell line (Yau 1979). The relevance of MDA toxicity tests conducted on whole cells or on animals for MDA generated in vivo is questionable. Although a relatively small dose of MDA (2 pg/g body weight) administered orally to rats results in the appearance of traces of free MDA in the urine (McGirr et al. 1985), little or no free MDA is detectable in normal human plasma, urine or solid tissues. A negligible fraction of MDA in foods is absorbed in the free form (PichC et al. 1988a) and hence it is unlikely that the diet is a significant source of free MDA in the tissues. The relative benignancy of MDA administered orally therefore may have little relevance to the toxicity of MDA formed, for example, in the nuclear membrane in proximity to the genome, by stimulation of lipid peroxidation by environmental factors. The effects of adding MDA to a culture medium for rat skin fibroblasts are listed in table 1 (Bird and Draper 1980, Bird et al. 1982a). Studies on [1,3-I4C]MDA metabolism indicated that, as a result of binding to proteins in the medium, MDA uptake was only 4%. The nuclear effects seen at concentrations of lo-' to M in the medium therefore may have occurred at intracellular concentrations as low as
Metabolism of malondialdehyde
903
Table 1 . Effects of adding MDA as the enol Na salt to the medium of rat skin fibroblasts grown in culture’. Treatment ~ O - ’ M , 120h
1 0 - 4 ~12h ,
Xenobiotica Downloaded from informahealthcare.com by National University of Singapore on 06/17/14 For personal use only.
1 0 - 4 ~120h , to 1 0 - 6 ~3h , to 1 0 - 6 ~120h ,
Effects Altered morphology, cytoplasmic vacuolization, karyorrhexis, micro- and multinucleation, decreased mitotic index, decreased DNA, RNA and protein synthesis Chromosomal fragments, achromatic lesions, chromatid breaks Mitotoc aberrations, micronucleation,decreased mitotic index, decreased DNA Increased DNA repair synthesis Small and irregular nuclei
’Bird and Draper 1980, Bird et al. 1982a. Studies on [1,2-’*C]MDA uptake showed that -95% of the radioactivity remained, bound to protein, in the medium.
~ O - ’ M . The material used in these cell culture studies, as well as in the animal studies cited below (Bird et al. 1982b, Siu et al. 1983), was purified by recrystallization three times from acetone and shown to be free of side-products (Marnett and Tuttle 1980) by chromatography on Sephadex LH-20.
Toxicity of oral M D A The acute oral LD,, of MDA for rats, administered as the enol Na salt (Na oxyacrolein), has been found to be 632pglg body weight (Crawford et al. 1965). Administration of MDA to mature Swiss mice at levels of 2,10, 50,250 or 500 pg/g body weight per day for 90 days resulted in no general histopathology (Siu et al. 1983). However, the liver nuclei exhibited dose-dependent irregularities including anisokaryosis, hyperchromicity and vesiculation, and at the highest level there was weight loss and atrophy of the exocrine cells of the pancreas with loss of zymogen granulation. Administration of MDA as the enol Na salt in the drinking water of Swiss mice at levels of 0.1,l and 10pg/g body weight per day for 12 months resulted in no increase in the incidence of lesions in 27 tissues examined, except for the liver (Bird et al. 1982 b). Hepatic abnormalities, observed at the two highest doses, included anisokaryosis, changes in cytoplastic volume with architectural derangements, necrosis, and neoplastic changes (nodular hyperplasia, hepatoma and haemangioma). There was no increase in specific neoplasms, but the incidence of total neoplasms and neoplastic lesions was dose-dependent (4%, 8% and 12%, respectively, vs. 1% in controls) (P
