Polycyclic Aromatic Hydrocarbons Evaluation of Sources and Effects (1983) / Chapter Skim
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5 EFFECTIVE BIOLOGIC DOSE
5 EFFECTIVE BIOLOGIC DOSE
Pages 207-272
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From page 207... ...
OH 0R . OH OH \ ~1, 2-Dlhydroxy 5-Me thylchrysene HO H3 OUCH ~7,8-Dihydro 5-Bydroxy-H >~ 9''10-Dlhydro-9, 10-tihydroxy FLUORENONE There appear to have been no direct studies on the metabolism of fluorenone~ but N-3-fluorenyl acetamide (3-FAA)
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From page 208... ...
- o 9-Fluorenone o l o ~ N-C-cH" H H N-3-fluorenyl acetamide ( 3 FM) }£E: THYLFLUORENE (~1 ~ LO I ~0 H 1 o 3~ ~C-Cu3 9~Bydroxy-3 FAA o 4~N-~-C H
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From page 209... ...
pyrene has been ident i f fed as a component of carbon black.66, 9 Its metabolism has been studied in rat-liver microsomes.67 The major metabolize isolated by HPLC has been identified as trane-3,4-dihydroxy-3,4-dihydrocyclopenta~ct~pyrene. Several came portents not yet well characterized consisted presumably of phenolic derivatives, as well as metabolites that appear to have saturation of the ethylene bridge.
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From page 210... ...
Enzymes other than microsomal monooxygenases may also be involved in the metabolic activation of PAHs. Eling et al.52 and Marnettl25 have shown that numerous xenobiotics, including the dihydrodiol metabolites of PAHs, can be cooxygenated during the oxygenation of arachidonic acid by prostaglandin synthetase.
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From page 211... ...
s Dibenzothiophene Benzo thiophene H OH OH ASH LC-H o .
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From page 212... ...
~ In addition, they are b li f pAHs70~72-74,104,163~170 and will bind to DNA in vitro.18 However, it became obvious that K-region epoxide-DNA adducts were not the adducts formed in viva between BaP metabolites and DNA.18 A similar conclusion was reached in studies with 7-MBA.17 Borgen et al.22 found that, in a microsomal activating system, the 7,8-diol BaP metabolite bound to DNA to a much greater extent than any other known dial or phenol. Sims et al.172 provided evidence that the BaP metabolite-DNA adduct formed by BaP metabolism by Syrian hamster-embryo cells in culture was chromatographically ~ dentical with an adduct formed by metabolism of BaP 7, 8-dio1 and proposed that t'ne reactive metabo1ite was a diol-epoxide (DE)
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From page 213... ...
Jerina and Daly94 pointed out that the epoxide ring is in a bay region and suggested the term "bay-region diol-epoxides" for these highly reactive metabolites of PAHs. The bay-rezion diol-enoxides are mutagenic 89,96,115,117,126,136,174,185,186 have transforming activity in mammalian cells,843126 are carcinogenic in newborn mice3l~l00,l0l~ll7 and Chinese ham te 1 in cells 1173187 a d are initiators in cells of mouse skin.~55~&0~0~,ii5~7~75 The mutagenicity and carcinogenicity, combined with the observation that bay-region diol-epoxide-DNA adducts are the major adducts formed in viva in target tissue, have led to the hypothesis that bay-region diol-epoxide adducts are the ultimate carcinogens generated by metabolism of most PAHs.963115 However, it should be pointed out that many nonmetabolized PAHs that are termed carcinogenic either lack a bay-region benzene ring or contain nonreactive substitutes in this molecular region.80 In addition, PAH metabolite-DNA adduct formation in viva has been examined only for BaP, DMBA, and 3-MC, and these studies have concentrated on target tissues in mice (see Table 5-1~.
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From page 214... ...
The only identified adducts observed by Sephadex LH20 chromatography were BaP DE-DNA adducts. Early-eluting radioactivity was present in the chromatograph.51 Eastman and Bresnick50 and Eastman et al.51 used Sephadex LH20 chromatography to analyze the 3-MC metabolite-DNA adduct profile in lung and liver of several mouse strains after intravenous administration of 3-MC (Figure S-7~.
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From page 215... ...
9-51,130 Sephadex LH20 chromatography of DMBA-deoxyribonucleoside adducts in skin was similar in each mouse strain studied; three peaks were observed.150 Early-eluting peaks were also observed in the chromatography of these investigations of PAR metabolite-DNA adduct formation in mouse skin. The formation of DNA adducts of the carcinogen 15,16-dihydro11-methylcyclopentataiphenanthrene-17-one (11-me2t3~1ketone)
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From page 216... ...
, in picomoles per milligram of DNA, of PAH metabolite-DNA adducts have been determined in several tissues after administration of PAHs. Table 5-2 gives the SAs of BaP DE adducts in lung and liver of various mouse strains and New Zealand rabbits.
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From page 217... ...
However, it is likely that the amounts of adducts formed in different cell types vary considerably. This possibility has the greatest implication for organs, such as the lung, that contain a multitude of cell types.
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From page 218... ...
Neither the amounts of PAH metabolite-DNA adducts nor their disappearance rates showed a correlation with the reported susceptibilities of the strains to PAH-induced skin carcinogenesis. Thus, the results of this study based on average cellular SAs of the organ are in agreement with the BaP, but not the 3-MC, study of pulmonary adducts in the different mouse strains.9~50390 Ashurst and Cohen 1 confirmed the results of Phillips _~4al.150 with HPLC analysis.
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From page 219... ...
. _ The effect of AHH inducers on the in vivo binding of BaP to DNA has been examined in several tissg~s of various mouse strains.36390~184 In a study by Wilson et al.,1 adducts were dete~uined under conditions known to resulE in inhibition of BaP-induced pulmonary 5-23 ~;
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From page 220... ...
The increase in total DNA-associated radioactivity was due to the increase in the amounts of the early-eluting peaks.36 Again, inhibition of BaP-induced tumor formation correlates with inhibition of BaP DE adduct formation in the target tissue, but not with total DNAassociated radioactivity on covalent binding of BaP to protein. In a continuation of their studies on the anticarcinogenic effects of TODD, DiGiovanni et al.44 showed that the time course for induction of epidermal AHH and UDPGT correlated with the time course for the inhibitory effects of TODD on tumor initiation with DMBA, BaP, and 3-MC.
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From page 221... ...
84 and Cohen et al.36 discussed above suggest that AHH inducers, such as OFF and TODD, inhibit BaP-induced neoplasia by reducing the amount of BaP DE-DNA adducts formed in the target tissue. The data of Pyerin et al.154 also suggest that prior AHH induction is a protective mechanism against DMBA-induced tumor initiation in mouse skin.
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From page 222... ...
In addition, in viva rates of disappearance of specific activities of adducts cannot be unequivocally equated with enzymatic excision rates, because cell turnover will also result in a decrease in the SA of the adduct. Eastman and Bresnick50 examined the disappearance of 3-MC metabolite-DNA adducts in lung and liver of several mouse strains (Table 5-4~.
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From page 223... ...
Similar conclusions were obtained with BaP and 3-MC, although adduct amounts were examined at only two points.150 Rayman and Dipplel56 examined the formation and disappearance of 7-bromomethylbenz~ajanthracene and 7-bromomethyl-12-methylbenz~aJanthracene metabolite-DNA adducts in skin of Swiss S mice. The adduct amounts of the less carcinogenic 7-bromomethylbenz~aJanthracene were higher and required a longer time to reach maximums -- 4 d vs.
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From page 224... ...
Although the extent of carcinogen-DNA binding has been successfully used to rank a series of carcinogens, such as PAHs and alkylating agents, for carcinogenic potency,30337388 there have been few, if any, serious attempts to use the amount of carcinogen-DNA adducts formed in the target tissue as a predictive tool for low-dose extrapolation in carcinogenesis. Anderson et al.7 and Gehring and Blau suggested a general scheme for the incorporation of pharmacokinetics in low-dose risk estimation for chemical carcinogenesis.
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From page 225... ...
Moreover, induced-mutation frequencies are linearly related, at least at sublethal doses of the mutagen, to the amounts of mutagen-DNA adducts present when the cells are undergoing replication. This linear relationship makes DNA adduct amounts a good measure of the effective biologic dose of mutagens in in vitro assay systems.
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From page 226... ...
Most in viva studies of carcinogen-DNA adduct formation have used total DNA content in an organ to calculate the specific activities of the adducts. However, it is likely that the amounts of adducts formed vary considerably in different cell types.
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From page 227... ...
~ Even if the initiation characteristics cannot explain organ, species, and strain differences, this does not detract from the use of PAN metabolite-DNA adduct amounts as a measure of the effective biologic dose of a carcinogen in a known target tissue. For low-dose extrapolation of carcinogenic data, for the ranking of a series of similar carcinogens, and for determining the effect on neoplasia of pretreatments that alter the metabolism of a carcinogen, the results discussed above strongly suggest that specific PAR metabolite-DNA adduct amounts are a good measure of effective biologic dose.
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From page 228... ...
Obviously, the amounts of DNA adducts constitute the appropriate measure of effective biologic dose for these · .
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From page 230... ...
- ~ cd to to to on en ~ ~ ~ ~ ~ ~ ~ ~If a ~ lo of ~ ~ ~ ~In ~ O ^ cO ~ ~ · e · ~ c' ¢ o E ·- ~ Z ~ U)
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From page 231... ...
TABLE 5-3 In Viva Formation of BP Metabolite-DNA Adduces in Lung and Liver of Rats Doseja Tissue ~mol/kg Specific Activity,b fmol/m~ of DNA Total DNA Binding BP-pheno 1-ox ideDNA Adduct . Lung 1.0133 + 11 55 e 5 + 6 ~ 0 Liver 1.0730 + 220 48.2 + 5.5 Lung 10.0 680 + 165 178 + 41 Liver 10 ~ 0 2 ~ 500 + 610 168 + 25 al.V.
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From page 232... ...
Values obtained by multiplying ~ of radioactivity eluting from Sephadex LH20 as peaks V and VI by total DNA-as soc iated radioac t ivity .
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From page 233... ...
Specific activity of BP DE-DNA adducts calculated from HPLC chromatogram. bMice were fed ~NF (3 mg/g of diet)
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From page 234... ...
BP Dose,a Strain mg/mouse T ime b h ? DNA TABLE 5-6 Effects of Pretreatment with TODD on In Vivo Covalent Binding of BP to Mouse Epidermal DNA, RNA, and Protein Hydrocarbon Bound to Macromolecules (pmol/mg)
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From page 235... ...
BP. Specific activity of BP DE-DNA adducts decreased monoexponentially in lung and liver of A/HeJ mice.
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From page 237... ...
- - ~ Chemico I ·xposilreExposure Uptoke ~Uptake 1 I Non m crow l :~1 enzymes 1 enzymes . _ ~_ Detoxification No ~ damage ~ Repolr Voicer.
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From page 238... ...
~ o~ ~ - ~o 0~ o >: at an l:' i to ...
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From page 239... ...
@; HOW on 't ~ of H0~ I)
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From page 240... ...
99~ HO"' fir FIGURE 5-S. Structures of BPDE; both isomers represent enant lamer ic mixtures .
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From page 241... ...
Boo lL c, IL c, w Nina We 5-45 O N O lo 1° .0 ___-,' O o o CO o TO Se ~ .° ~ o 0 0 ~0 2 ~ o 0 0 Pa C)
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From page 242... ...
~or Al ~ i'i'260 !
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From page 243... ...
An o 30 2 ,- - ~ to o ~ / / 1= ~ an 1& ~ Mo! DMSA/Mouse FIGURE 5-8.
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From page 244... ...
Dose dependence of BaP adduc t format ion in epidermal DNA. Groups of mice, 15-20 each were treated topically with 1, 2, 5, 10, 25, and 100 ng of []
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From page 245... ...
\ A: l o ~ En Is lo LL \ \ cr lo \ 8 \ \ ZJ : ~
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From page 246... ...
looo 750 500 250 : _ TA100 TABS / / so ° 5Yl-I · BP 9- phenol 100 200 30G No ElP Molecuics / Solmonel~a Genome FIGURE 5-11. Linear relation between BP DE I, BP DE II, and 9-hydroxybenzo~a~pyrene-induced mutation frequencies at histidine locus in Salmonella typhimurium strains TA 100 and TA 98 and BP metabolite-ONA adducts in bacterial cells.
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From page 247... ...
Crew. Repair of DNA adducts of the carcinogen 15,16-dihydro-11-methylcyclopentata~phenanthren-17-one in mouse tissues and its relation to tumor induction.
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From page 248... ...
Metabolism of benzo~a~pyrene and identification of the major benzo~a~pyrene-DNA adducts in cultured human colon. Cancer Res.
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From page 249... ...
Evidence for the binding of poly nuclear aromatic hydrocarbons to nucleic acid of mouse skin: Relation between carcinogenic power of hydrocarbons and their binding to deoxyribonucleic acid. Nature (London)
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From page 250... ...
Excision of 7-bromomethylbenz~aJanthracene-DNA adducts in replicating mammalian cells. Biochemistry 16:1499-1503, 1977.
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From page 251... ...
Cerutti. Excisability and persistence of benzo~a~pyrene DNA adducts in epithelioid human lung cells.
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From page 252... ...
Sims. The involvement of a diol-epoxide in the metabolic activation of benzo~a~pyrene in human bronchial mucosa and in mouse skin.
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From page 253... ...
A bacteriophage system for screening and study of~biologically active polycyclic aromatic hydrocarbons and related compounds.
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From page 254... ...
The bay-region theory of carcinogenesis by polycyclic aromatic hydrocarbons, ppe 173-188.
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From page 255... ...
The bay region theory of polycyclic aromatic hydrocarbon-induced carcinogenicity, pp.
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From page 256... ...
Sims. The metabolism of a series of polycyclic hydrocarbons by mouse skin maintained in short-term organ culture.
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From page 257... ...
Dipple. Metabolic activation of the carcinogen 7,12-dimethylbenz~aJanthracene for DNA binding.
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From page 258... ...
Sims. The covalent binding of polycyclic hydrocarbons to ONA in the skin of mice of different strains.
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From page 259... ...
Structure and activity in chemical carcinogenesis. Comparison of the reactions of 7-bromomethylbenz~aJanthracene and 7-bromomethyl-12-methylbenz~aJanthracene with mouse skin deoxyribonucleic acid in vivo.
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From page 260... ...
Calvin. Identification of the major adducts formed by reaction of benzo~a~pyrene diol epoxide with DNA ~n vitro.
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From page 261... ...
Interactions between polycyclic aromatic hydrocarbons and cellular macromolecules, pp.
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From page 262... ...
The major adducts of cis and bans benzo [a] pyrene diol epoxides cause chain termination during DNA synthesis in vitro.
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From page 263... ...
SKIN That benzota] pyrene hydroxylase can be induced in cultured human skin was first demonstrated in l972.1° Foreskins from children who were circumcised 2-4 d after birth were shown to contain an enzyme that bydroxylates the carcinogen benzo~aipyrene (BaP)
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From page 264... ...
In isolated cultured human hair follicles, Vermorken _ al.,185 using radioactive BaP as substrate, not only demonstrated the presence of the hydroxylase, but identified the formation of the 3-OH, 7,8-dihydro-7,8-dihydroxy, and 9,10-dihydro9,10-dihydroxy metabolites of this PAH. BaP clearly has cytotoxic effects on cultured human skin fibroblasts, although relatively high concentrations are required for cytotoxicity.
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From page 265... ...
Two principal BaP-DNA adduct peaks could be identified -- a major peak consistent with an adduct standard synthesized from the anti-isomeric 7,8-dibydrodiol-9,10-epoxide of the hydrocarbon and a minor peak consistent with the syn-isomeric form of this metabolize. The results are consistent with those in other reports on BaP adducts formed in human explant tissue from lung,161 colon,l° esophagus,68 and bronchus,75 and they represent an advance in the development of sensitive assay systems for detecting biologic responses to human epithelial-cell activation of BaP.
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From page 266... ...
Such parallelism is of more than casual interest, in view of the susceptibility of the mouse to hydrocarbon carcinogenesis and the known correlation between hydrocarbon-DNA binding and cancer-producing activity in mouse skin. The effects of pyrene and BaP in the human diploid fibroblast culture WI-38 were studied by Weinstein et al.188 Neither caused significant damage (compared with controls)
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From page 267... ...
Some of the PAH-deoxyribonucleoside adducts formed from 7,12-DMBA and BaP appeared to have been produced through reactions of bay-region diol-epoxides with DNA, but little reaction with DNA was detected in tissue preparations treated with BaP. Unscheduled DNA synthesis induced by DNA-damaging chemicals has been measured in nonreplicating human fibroblasts by autoradiographic methods that are not readily applicable to organotypic epithelial-cell cultures.
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From page 268... ...
Dybing et al.54 have examined the in vitro metabolism and metabolic activation of several carcinogenic PAHs in subcellular fractions from seven human livers. The patients all suffered from total cerebral infarction and were serving as potential kidney donors (maintained temporarily by life-support systems)
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From page 269... ...
Pretreatment of rats with unlabeled BaP greatly increased the plasma disappearance rate of a tritiated dose of the same compound given intravenously; the effect was especially marked during the first 5 min after the intravenous dose of the radiolabeled material, and the increased rate lasted for at least 6 h. This effect of pretreatment with the compound was paralleled by a lower concentration of [3H]
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From page 270... ...
A greater proportion of benzene-ring metabolites was formed by human lung microsomes than by human liver and kidney microsomes (or rodent lung microsomes)
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From page 271... ...
Other evidence that these catalytic sites are different has also recently been provided by the studies of Yoshinaga et al.197-200 Metabolic transformation of PAHs and their binding to cellular macromolecules in cultured human gut tissues have been described. Harris et al.68 examined the metabolic fate of BaP and several other compounds in cultured esophogeal explants from eight patients, six of whom had esophageal carcinomas.
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From page 272... ...
A number of hydrocarbon-DNA adducts were identified, and both qualitative and quantitative rat-human differences were demonstrated. Although overall BaP metabolism was similar in rat and human colon tissue, the ratio of organic-solvent-soluble to water-soluble metabolites was higher in the human; sulfate esters predominated in rat colon, whereas equivalent quantities of sulfate esters and glutathione conjugates were formed in the human tissue; and hydrocarbon-DNA binding was distinctly greater in human colon, although, as noted, there was marked variability in adduct formation within a given subject.
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