The DNA binding of benzo[alpha]pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated perfused rat lung.

When [³H]benzo[a]pyrene is incubated in vitro together with DNA, NADPH and rat lung microsomes, covalent binding of benzo[a]pyrene (BP) metabolites to DNA occurs. These metabolite-nucleoside complexes can be resolved into several distinct peaks by elution of a Sephadex LH-20 column with a water-methanol gradient. 3-Methylcholanthrene (MC) pretreatment of animals induces the total covalent binding in vitro several-fold and increases the amounts of at least five metabolite-nucleoside complexes associated with the 7,8-diol-9,10-epoxidcs, the 7,8-oxide or quinones oxygenated further, the 4,5-oxide and phenols oxygenated further. These increases correspond well with the increases in the production of both non-K-region and K-region metabolites of BP by lung microsomes, as determined by highpressure liquid chromatography (HPLC). On the other hand, when [³H]BP is metabolized in isolated perfused rat lung, only the peak representing the 7,8-diol-9,10-epoxide bound to nucleoside(s) is readily detectable and then only in lungs from MC-treated animals. The extent of binding of BP metabolites to lung DNA is very low, about 0.0004% of the total dose applied to the perfusion medium; more than 60% of this can be accounted for by the binding of the 7,8-diol-9,10-epoxides to nucleoside(s). It is suggested that the further metabolism leading to metabolites not available to covalent binding, (e.g. conjugation) of primary BP metabolites in the intact tissue is responsible for the differences in the metabolite-nucleoside patterns observed in vivo, as compared with microsomal metabolism in vitro.     

Nuclear metabolism of benzo(a)pyrene and of (±)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene: Comparative chromatographic analysis of alkylated DNA

Rat liver nuclei were incubated with [¹⁴C]benzo(a)pyrene (BP) or [³H](±)-trans-7,8-dihydrodiol of BP (³H-BP-7,8-diol) in the presence of a NADPH-generating system. The nuclei were able to form from BP the 9,10-, 4,5- and 7,8-dihydrodiols, the 3,6- and 1,6-quinones as well as the 3- and 9-phenols. The total nuclear metabolism was stimulated 11-fold by prior administration to the rats of 3-methylcholanthrene (3MC). BP-7,8-dihydrodiol formation, under these circumstances, was enhanced 29-fold. The rat liver nuclei were also able to form from [³H]BP-7,8-diol, (±)-7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 1), (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 2), as well as three unknown metabolites. Diol epoxides 1 and 2 represented 23 and 65% of the total metabolites produced during the control nuclear incubation. Pretreatment of the rats with 3MC resulted in 4-fold increase in nuclear metabolic activity. Under the latter circumstances, the diol epoxides 1 and 2 represented 43 and 38%, respectively, of the total nuclear metabolites. Incubation of liver nuclei with labeled BP or BP-7,8-diol in the presence of NADPH resulted in alkylation of DNA. The alkylated deoxyribonucleosides were separated by Sephadex LH-20 chromatography. Two peaks of radioactivity were noted after incubation with the parent polycyclic hydrocarbon while only one peak was seen after incubation with the diol derivative. These results emphasize the importance of nuclei in the metabolism of BP and in the subsequent alkylation of DNA, reactions which may be related to mutagenesis or carcinogenesis.     

Uptake of bacterial DNA by Chlamydomonas reinhardi

Escherichia coli [³H]DNA supplied to vegetative cultures of wild-type (mt+) and CW15 (mt+; mutant lacking the cell wall) Chlamydomonas reinhardi could bind to the cell wall of the wild-type and to the cell membrane of CW15 mutant cells. The extent of this binding decreased with time and was to a large degree (over 90%) DNA-ase-sensitive. Nevertheless, about 0.01% of the bacterial DNA remained irreversibly associated with the cells when they reached stationary phase. The irreversible binding of the donor bacterial DNA to Chlamydomonas cells could be increased by treatment of the cultures with polycations such as DEAE-dextran, poly-L-lysine and poly-L-ornithine. Although the CW15 cells rapidly degraded bacterial DNA in the culture medium wild-type cells showed only a small effect on the molecular weight of the donor DNA.The acid-insoluble radioactivity irreversibly bound to WT (+) cells consisted mainly of oligonucleotides with a small proportion present as less depolymerized donor DNA. No radioactivity, however, was found to be associated with the recipient high molecular weight Chlamydomonas DNA.No labeled donor DNA could be recognized in the cells given bacterial [³H]DNA in early stationary phase. Instead, radioactivity found in Chlamydomonas DNA corresponded to reutilization of [³H]thymine derivatives released as a result of [³H] DNA degradation. No evidence for the integration of detectable amounts of donor DNA sequences into the host cell DNA was obtained.     

The interaction of carbon-14-labelled alkyl carbamates, labelled in the alkyl and carbonyl positions, with DNA in vivo

The binding of ethyl carbamate labelled with carbon-14 in the alkyl or carbonyl group, and of methyl, n-butyl and n-propyl carbamates labelled in the alkyl group, to the DNA of mouse liver, lung and kidney has been studied in male Crackenbush mice. Only ethyl carbamate bound to liver and kidney DNA to any significant extent.The binding of ethyl carbamate labelled with carbon-14 in the C₁, C₂ or the carbonyl position was examined and compared. The levels of binding of [1-¹⁴C]- and [2-¹⁴C]ethyl carbamate to liver DNA were not significantly different (328 ± 34 and 267 ± 24 dpm/mg DNA, respectively), but there was very little binding of the [carbonyl-¹⁴C]ethyl carbamate (26 ± 3 dpm/mg DNA). Furthermore, only 18% of the radioactivity was removed from the DNA labelled with the alkyl-labelled carbamates, whereas 65% of the radioactivity was removed from the DNA labelled with carbonyl-labelled ethyl carbamate on continuous ether extraction. It was concluded that the bound molecule does not contain the carbonyl carbon and is probably an ethyl group.     

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.     

Microsomal activation of thioacetamide-S-oxide to a metabolite(s) that covalently binds to calf thymus DNA and other polynucleotides

In the presence of NADPH liver microsomes isolated from phenobarbital-pretreated rats catalyze the conversion of [³H]thioacetamide-S-oxide to a reactive intermediate(s) which covalently binds to calf thymus DNA, calf liver RNA, polyguanylic acid (poly(G)) and polyadenylic acid (poly(A)). The highest level of binding of radioactivity was obtained with poly(G), followed by poly(A), RNA and DNA. The incorporation of radioactivity into DNA was linear for 30 min and there was a requirement for NADPH for time-dependent covalent binding to occur. Performing the microsomal incubations in an atmosphere of 80% CO/20% O₂ or adding partially purified anti cytochrome P-450 immune serum to the microsomal incubations inhibited the total metabolism of thioacetamide-S-oxide and had a small, but insignificant, inhibitory effect on binding of radioactivity to calf thymus DNA. Using a reconstituted monooxygenase system containing cytochrome P-450 purified from phenobarbital-treated rats we were unable to detect any metabolism of thioacetamide-S-oxide. Only background levels of radioactivity were incorporated into calf thymus DNA when microsomes isolated from phenobarbital-treated rats were incubated with [³H]thioacetamide in the presence of NADPH. These results suggest that thioacetamide-S-oxide is an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s) which binds to calf thumus DNA.     

Synthesis and evaluation of [11C]cyanoimipramine

[¹¹C]Cyanoimipramine has been prepared by methylation of the desmethyl cyanoimipramine with [¹¹C]methyl iodide. The chemically and radiochemically pure labelled product was obtained with a high specific activity (> 300 mCi/μmol). When ¹¹C (or ³H)-cyanoimipramine was intravenously administered in mice, high accumulations were shown in brain and lung. Thirty minutes after injection of the tracer, differences were found in the radioactivity between the cerebral cortex and the cerebellum. The regional distribution of radioactivity in the rat brain 30 min after i.v. injection of [¹¹C]cyanoimipramine was also examined, and the radioactivity was high in receptor rich areas (striatum, cerebral cortex etc.) but low in receptor poor area (cerebellum). The in vivo stability of [³H]cyanoimipramine was quite stable in the mouse brain for at least 30 min. Thirty minutes after injection, the radioactivity in the cerebral cortex of the carrier-added state was reduced as compared with the carrier-free state. Taken together, the in vivo specific binding of [³H]cyanoimipramine in the cerebral cortex was estimated at about 40–50% of the total radioactivity. Furthermore, the distribution of [³H]cyanoimipramine in the mice forced to swim was examined. Significant changes in the distribution of [³H]cyanoimipramine were observed in the cerebral cortex.     

Metabolism of benzo[a]pyrene and persistence of DNA adducts in the brown bullhead (Ictalurus nebulosus)

• 1.1. The in vitro metabolism of [³H]benzo[a]pyrene (BP) and [¹⁴C]benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) by liver of brown bullhead (Ictalurus nebulosus) was characterized, as was the formation and persistence of BP-DNA adducts in vivo.
• 2.2. Compared to rat liver microsomes, bullhead liver microsomes produced relatively larger amounts of BP-7,8-diol (predominantly the [−] enantiomer) and smaller amounts of BP-4,5-diol.
• 3.3. BP phase I metabolites were efficiently converted by freshly isolated bullhead hepatocytes to conjugates, predominantly glucuronides.
• 4.4. BP-7,8-diol was metabolized by hepatocytes 4-fold more rapidly than was BP and was converted to approximately equal amounts of glucuronides, glutathione conjugates and sulfates.
• 5.5. BP-DNA adducts formed in bullhead liver with a lag time of several days and maximum adduct formation at 25–30 days. The major adduct was anti-BPDE-deoxyguanosine.

     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Covalent binding of ethinylestradiol and estrone to rat liver DNA in vivo

The covalent binding of [6,7-³H] ethinylestradiol (EE) and [6,7-³H] estrone (E) to liver DNA of 200 g female rats was measured 8 h after the administration of 80 μg (9.2 mCi) estrogen by gavage. The binding is 1.5 for EE and 1.1 for E, expressed as binding to DNA/dose, in units of μmol hormone/mol DNA phosphate/mmole hormone/kg body wt. It is in the same order of magnitude as for benzene and about 10 000 times below the binding of typical liver carcinogens, such as aflatoxin B₁ or N,N-dimethylnitrosamine.     

Ubiquitous binding of benzo[a]pyrene metabolites to DNA and protein in tissues of the mouse and rabbit

The in vivo formation of benzo[alpha]pyrene (BP) metabolite-DNA adducts in several tissues of mice and rabbits was examined. Included were tissues with widely divergent xenobiotic metabolizing capabilities such as liver and brain. The major adduct identified in each tissue was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDEI)-deoxyguanosine adduct. A 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BP (BPDEII)-deoxyguanosine adduct, a (-)-BPDEI-deoxyguanosine adduct and an unidentified adduct were also observed. These adducts were present in all of the tissues of the mice and in the lungs of the rabbits; only BPDEI and BPDEII were seen in the rest of the rabbit tissues. In all of the tissues studied, the DNA adduct levels were unexpectedly similar. For example, the BPDEI-DNA adduct levels in muscle and brain of mice were approx. 50% of those in lung and liver at each oral BP dose examined. After an i.v. dose of BP in rabbits, the BPDEI adduct levels in lung were three times those in brain or liver and twice those in muscle. The binding of BP metabolites to protein was also determined in these tissues. The tissue-to-tissue variation in protein binding levels of BP metabolites was greater than that for BPDEI-DNA adducts. There are several possible explanations for the in vivo binding of BP metabolites to DNA and protein of various tissues. First, oxidative metabolism of BP in each of the examined tissues might account for the observed binding. Second, reactive metabolites could be formed in tissues such as liver and lung and be transported to cells in tissues such as muscle and brain where they bind to DNA and protein. In any case, the tissue-to-tissue variations in protein and DNA binding of BP-derived radioactivity do not correlate with differences in cytochrome P-450 activity.     

Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in cultured fibroblasts

Nucleotides and sugar nucleotides were extracted from cultures of human fibroblasts with perchloric acid, separated by isotachophoresis, and quantified by uv absorption analysis at 254 nm. ATP (936 pmol/μg DNA) was, as expected, the dominating nucleotide pool. The energy charge was estimated to 0.9. The UDP-N-acetylhexosamine pool was also a very prominent compound (596 pmol/μg DNA). After incubation of fibroblasts with [³H]glucosamine, more than 95% of the acid-soluble radioactivity was found in the UDP-N-acetylhexosamine pool. Incubation with [³⁵S]sulfate resulted in the incorporation of [³⁵S]sulfate into 3′-phosphoadenosine-5′-phosphosulfate (PAPS). The latter could, however, only be measured as radioactivity, as the amount was too small to be quantified as total mass. Pulse-labeling of fibroblasts with [³⁵S]sulfate and [³H]glucosamine from 5 min to 16 h showed that [³⁵S]PAPS was equilibrated in less than 10 min, while [³H]glucosamine required a longer time, 2–4 h, to attain a steady state with UDP-N-acetylhexosamine. [¹⁴C]Glucose required approximately the same time as [³H]glucosamine to reach steady state with UDP-acetylhexosamine, which suggests that the reason for the long equilibration time is the slow turnover of this pool.     

Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver

The distribution of radioactivity among pyrimidine isostichs (or isoplyths) of DNA from 24-h regenerating rat liver was studied with [³H]Thd, [¹⁴C]orotate or with inorganic ³²P_i. Expression of incorporated radioactivity as log₁₀% of total radioactivity recovered for each of the 11 pyrimidine isostichs detected showed that radioactivity from [³H]Thd was asymmetrically distributed among the isostichs, i.e., ³H radioactivity failed to access regions of DNA yielding lower molecular weight pyrimidine isostichs as efficiently as it accessed regions yielding higher molecular weight pyrimidine isostichs. The thymine (T) content of isostichs exceeded that of cytosine (C), i.e., ratios for the first 10 isostichs averaged 1.43 ± 0.08 and 1.28 ± 0.05, depending on the method of analysis; furthermore, the ratio for isostich 1 was significantly higher than ratios for isostichs 2 through 10. Asymmetric distributions of [³H]Thd radioactivity also were seen at 18 or 30 h post-partial hepatectomy. Thus, radioactivity from [³H]Thd, a DNA precursor from the salvage pathway, failed to efficiently access lower molecuar weight isostichs despite thymine enrichment, suggesting that thymine moieties were supplied from additional sources. Radioactivity from [¹⁴C]orotate accessed lower molecular weight pyrimidine tracts more efficiently than [³H]Thd, but less efficiently than it accessed higher molecular weight isostichs, resulting in an asymmetric distribution of ¹⁴C radioactivity. This result suggested that appreciable quantities of thymine and cytosine moieties utilized for DNA synthesis were supplied de novo, but other sources also were utilized. Radioactivity from ³²P_i, a de novo precursor, was distributed symmetrically, i.e., the slope among lower molecular weight isostichs increased enough that it was indistinguishable from slopes for intermediate and higher molecular weight isostichs. Since ³²P radioactivity among lower molecular weight isostichs reflects appreciable contributions of de novo phosphate moieties from both pyrimidine- and purine-containing deoxynucleoside triphosphates, opportunities for observing contributions of ³²P radioactivity from pathways other than the de novo pathways appeared to lie beyond limits of detectability. The distribution of radioactivity from labeled DNA precursors among lower molecular weight pyrimidine tracts (a) indicate that thymine moieties are contributed by both salvage and de novo pathways; (b) support the possibility that cytosine moieties also are contributed by both pathways; and (c) support the ‘replitase’ concept for channeling dNTPs to replicating forks.     

Lack of contribution of covalent benzo[a]pyrene-7,8-quinone–DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0 mg/kg. Lungs and livers were harvested after 24 h, the DNA extracted and subjected to ³²P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50 mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis.     

Mechanisms of DNA Utilization by Estuarine Microbial Populations

The mechanisms of utilization of DNA by estuarine microbial populations were investigated by competition experiments and DNA uptake studies. Deoxyribonucleoside monophosphates, thymidine, thymine, and RNA all competed with the uptake of radioactivity from [³H]DNA in 4-h incubations. In 15-min incubations, deoxyribonucleoside monophosphates had no effect or stimulated [³H]DNA binding, depending on the concentration. The uptake of radioactivity from [³H]DNA resulted in little accumulation of trichloroacetic acid-soluble intracellular radioactivity and was inhibited by the DNA synthesis inhibitor novobiocin. Molecular fractionation studies indicated that some radioactivity from [³H]DNA appeared in the RNA (10 and 30% at 4 and 24 h, respectively) and protein (approximately 3%) fractions. The ability of estuarine microbial assemblages to transport gene sequences was investigated by plasmid uptake studies, followed by molecular probing. Although plasmid DNA was detected on filters after filtration of plasmid-amended incubations, DNase treatment of filters removed this DNA, indicating that there was little transport of intact gene sequences. These observations led to the following model for DNA utilization by estuarine microbial populations. (i) DNA is rapidly bound to the cell surface and (ii) hydrolyzed by cell-associated and extracellular nonspecific nucleases. (iii) DNA hydrolysis products are transported, and (iv) the products are rapidly salvaged into nucleic acids, with little accumulation into intracellular nucleotide pools.     

The interaction of ( 3 H)ethyl carbamate with nucleic acids of regenerating mouse liver

The binding of [³H]urethane to liver DNA and RNA has been examined in partially hepatectomised and intact male Crackenbush mice. A single dose of [³H]urethane (50 μCi) was given to non-hepatectomised mice (group A) and to 3 groups of partially hepatectomised mice at 18 (B), 28 (C) and 38 (D) hours postoperatively, respectively. The binding was examined over the subsequent 16 h. The maximum levels of binding to DNA declined in the order, group A > B > C > D, although the binding to DNA persisted longest in group B. The binding to RNA was greater in groups B, C and D than in group A. Neither the restoration of liver mass nor an alteration in the metabolism of urethane appeared to account for the different levels of binding. In normal and partially hepatectomised mice a single dose of urethane (20 mg) was followed by an inhibition of mitosis and of the incorporation of [³H]thymidine into liver DNA and of [³H]uridine into liver RNA.     

Pharmacokinetics,Tissue Distribution and Metabolism of Intravenously Administered Digalactosyldiacylglycerol and Monogalactosyldiacylglycerol in the Rat

Abstract

A bilayer forming galactolipid, digalactosyldiacylglycerol (DGalDG) has been identified as a tool with suitable physicochemichal properties for pharmaceutical formulation work. One possible application is as a carrier for liposome entrapped drugs for intravenous administration. The fate of intravenously administered galactolipids is not known. In this study liposomal dispersions of galactolipids, containing [³H]fatty acid labelled DGalDG or monogalactosyldiacylglycerol (MGalDG) were injected intravenously in the rat and the disappearance from blood and uptake by tissues were examined. The T1/2 of [³H]DGalDG in plasma was 3 to 5 minutes. Of the tissues examined (liver, spleen, kidneys, lung, heart, stomach, upper and lower small intestine and colon), the liver contained the highest radioactivity per g tissue after both 15 min. and 4 h. Autoradiographic examinations after 15 min, 1 h and 4 h showed that the uptake of radiolabeled DGalDG and MGalDG occurred mainly to the hepatocytes. Less than 6 % of the injected [³H]DGalDG remained in liver and plasma as [³H]DGalDG after 4 h. [³H]MGalDG exhibited a similar pattern of metabolism although the initial disappearance rate was faster than for [³H]DGalDG. The study thus shows that the hepatocytes take up and hydrolyse galactolipids after intravenous administration.     

Metabolism of benzo(a)pyrene by isolated hepatocytes and factors affecting covalent binding of benzo(a)pyrene metabolites to DNA in hepatocyte and microsomal systems

Covalent binding of benzo(a)pyrene (BP) metabolites to DNA was investigated in hepatocytes and liver microsomes (MC-microsomes) isolated from 3-methylcholanthrene-treated rats. The major DNA adducts formed during BP metabolism in both hepatocytes and incubations of calf thymus DNA with MC-microsomes were adducts of anti and syn isomers of trans-7,8,-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol-epoxides) and of epoxide derivatives of BP-9-phenol (phenol-oxides). Diol-epoxide adducts predominated over phenol-oxide adducts in hepatocytes, while the reverse was found in microsomal incubations. In hepatocytes, both diol-epoxide and phenol-oxide adducts increased with increasing BP concentration; the ratio of diol-epoxide adduct to phenol-oxide adduct decreased from 6:1 to 3:1 between 30 and 100 μm BP. In microsomal incubations, decreases in DNA concentration or addition of the hepatocyte L15 medium produced larger decreases in phenol-oxide adducts than in diol-epoxide adducts. The effects of the inhibitors salicylamide, diethylmaleate, and 3,3,3,-trichloropropene oxide on formation of BP-DNA adducts are interpreted in terms of changes in precursor formation and metabolism and reductions in hepatocyte glutathione levels. Addition of 1.5 mg/ml exogenous DNA to hepatocyte incubations produced no change in covalent binding to cellular DNA, even though extracellular BP-DNA adducts accounted for 97% of the total adducts formed. Both the relative amounts of diol-epoxide and phenol-oxide adducts and the total adducts per milligram of DNA were indistinguishable with respect to extracellular and intracellular DNA. Modification of extracellular DNA by diol-epoxides was at least as efficient as modification of calf thymus DNA in incubations with MC-microsomes. It is concluded that BP diol-epoxides and phenol-oxides can leave the cell or enter the nucleus with equal facility but are more effective in binding to DNA in the cell in which they are generated.     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

Thiolytic cleavage of the anti-tumour compound 4′-(9-acridinylamino)-methanesulphon-m-anisidine (m-AMSA,NSC 156 303) in blood

4′-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA), a compound with a broad spectrum of experimental anti-tumour activity, was found to have a short biological half-life in mice bearing L1210 leukaemia. The fate of m-AMSA [³H]-labelled in the acridine nucleus, was determined following injection into mice. There was rapid formation of covalent adducts with plasma proteins. Adducts were also formed in freshly isolated blood samples following incubation at 37°C, and were found to be highly fluorescent. The formation of adducts was accompanied by a decrease in the free thiol concentration in plasma, and the concomitant addition of radioactivity from [³H]acridine nuclei. Acid or alkaline hydrolysis of the plasma protein adduct liberated acridone, while digestion with a protease produced unstable fluorescent compounds. A comparison of the rates of acid hydrolysis of the adducts and of model compounds suggested that the adducts were produced as a result of nucleophilic attack at the C-9 position of m-AMSA by protein thiol groups. The side chain of m-AMSA was liberated as 4-amino-3-methoxymethanesulphonanilide. Several congeners of m-AMSA were shown to form similar or identical adducts both in vivo and in vitro, and at rates which correlated with their reactivity towards simple organic thiols.