ß-Catenin overexpression causes an increase in inflammatory cytokines and NF-κB activation in cardiomyocytes.

ß-Catenin has been implicated in various developmental and physiological processes. Defective Wnt signaling can result in different cardiac and vascular abnormalities and is activated under pathological conditions such as inflammation and obesity. In this study, roles of ß-catenin in inflammation in cardiomyocytes were investigated. 10 samples from hearts of patients with acute infarction and 10 from normal ones were collected in order to access roles of ß-catenin in cardiomyocytes. H9c2 cardiomyoblasts and primary neonatal rat cardiomyocytes were transfected with porcine cytomegalovirus (pCMV)-ß-catenin plasmid in order to overexpress ß-catenin. Protein level of ß-catenin protein was increased in human acute infarction tissues compared to ones from normal patients. The transcription factor had increased nuclear localization in cardiomyocytes of the Wistar rats with cardiac hypertension. Furthermore, expression of fibrosis protein markers increased. Protein expression of ß-catenin was increased in human acute infarction inflammatory heart tissues and in hearts of inflammatory obesity rats. After pCMV-ß-catenin plasmid was transfected in a dose-dependent manner, inflammation protein markers, TNF-α and IL-8, were upregulated in hypertensive neonatal rat cardiomyocytes and H9c2 cardiomyoblasts. In addition, overexpression of ß-catenin induced activation and nuclear localization of NF-κB. Therefore, ß-catenin is a potential molecular target for treatment of inflammation and fibrosis in cardiomyocytes.

Reperfusion using lactate Ringer's mixture partially eliminates IGF II receptor involved cardiac damage caused by hemorrhagic shock in diabetic rats.

Diabetes is a metabolic disorder that damages many organs. We investigated the effects of reperfusion using lactate Ringer's solution (LR) in a diabetic animal model. Eight-week-old rats were divided into groups: control, hemorrhagic shock induced (HS), diabetes mellitus (DM), DM plus HS (DM + HS) and DM rats that received LR after HS (DM + HS + LR). HS was induced by withdrawing blood from the femoral artery and arterial pressure was maintained at 40 mm Hg for 1 h. Animals were perfused with either withdrawn blood or LR. Rats were sacrificed and hearts were collected from all groups. Histopathological studies were performed using left ventricles and western blotting analysis was performed using protein extracted from the left ventricle. Using the TUNEL assay, we found more apoptotic cells in the DM + HS group compared to the control group, whereas in animals resuscitated with LR, the number of apoptotic cells was reduced. Western blotting showed a significant reduction in apoptotic markers, cyt c, cas 9 and cas 3, and increased survival markers, pPI3K and pAKT, in the DM + HS + LR group. Reperfusion with LR may have therapeutic effects on trauma induced HS by blocking the IGF II R facilitated apoptosis pathway in diabetic rats.

Permselectivity of of the glomerular capillary wall. Studies of experimental glomerulonephritis in the rat using neutral dextran.

Polydisperse [3h] dextran was infused into eight Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN), thereby permitting direct measurements of pressures and flows in surface glomeruli and fractional clearances for dextrans [(U/P) dextran/(U/P) inulin] ranging in radius from 18 to 42 A. Despite glomerular injury, evidenced morphologically and by a marked reduction in the glomerular capillary ultrafiltration coefficient, the glomerular filtration rate remained normal because of a compensating increase in the mean net ultrafiltration pressure. In NSN rats, as in normal controls, inulin was found to permeate the glomerular capillary wall without measurable restriction, and dextrans were shown to be neither secreted nor reabsorbed. For dextran radii of 18, 22, 26, 30, 34, 38, and 42 A, (U/P) dextran/(U/P) inulin in NSN and control rats, respectively, averaged 0.90 vs. 0.99, 0.81 vs. 0.97, 0.63 vs. 0.83, 0.38 vs 0.55, 0.20 vs. 0.30, 0.08 vs. 0.11, and 0.02 vs. 0.03. Using a theory based on macromolecular transport through pores, the results indicate that in NSN rats, effective pore radius is the same as in controls, approximately 50 A. In NSN, however, the ratio of total pore surface area to pore length, a measure of the number of pores, is reduced to approximately 1/3 that of control, probably due to a reduction in capillary surface area. These results suggest that proteinuria in glomerular disease is not due simply to increases in effective pore radius or number of pores, as previously believed. Using a second theoretical approach, based on the Kedem-Katchalsky flux equations, dextran permeability across glomerular capillaries was found to be slightly lower, and reflection coefficient slightly higher in NSN than in control rats.

Permselectivity of the glomerular capillary wall. Studies of experimental glomerulonephritis in the rat using dextran sulfate.

To determine whether the increased filtration of serum proteins after glomerular injury is the consequence of altered electrostatic properties of the glomerular capillary wall, we measured fractional clearances of the anionic polymer, dextran sulfate, in nine Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN). In agreement with previous studied from this laboratory, whole kidney and single nephron glomerular filtration rates were normal in NSN rats despite histological evidence of glomerular injury, and despite a marked reduction in the glomerular capillary ultrafiltration coefficient to approximately one-third of normal. In the companion study (9), it was shown that in NSN rats the mean fractional clearances of neutral dextrans over the range of effective molecular radii from 18 to 42 A were reduced, compared to normla. In contrast, in the present study the mean fractional clearances for dextran sulfate over the same range of molecular radii were significantly greater than those found previously for normal Munich-Wistar rats. The fractional clearance of dextran sulfate molecules of the same molecular radius as serum albumin (approximately 36 A) was increased markedly, from 0.015 +/- 0.005 (SEM) in nonnephritic controls to 0.24 +/- 0.03 in NSN (P less than 0.001). The sialoprotein content of glomeruli, estimated by the colloidal iron reaction, was reduced in NSN rats as compared to normal controls. It is concluded that the abnormal filtration of anionic serum proteins, such as albumin, seen in glomerulopathies is, at least in part, the consequence of loss of fixed negative charges from the glomerular capillary wall.

Mouse skin tumor-initiating activity of 5-, 7-, and 12-methyl- and fluorine-substituted benz[a]anthracenes.

As an approach for elucidation of structure activity relationships that underlie the exceptionally large difference in carcinogenic activity between benz[a]anthracene and 7,12-dimethylbenz[a]anthracene (7,12-DMBA), 11 methyl- and/or fluorine-substituted benz[a]anthracenes were evaluated for tumor-initiating activity on mouse skin. Outbred CD-1 and outbred Sencar mice received a single topical application of the hydrocarbons followed by twice weekly applications of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate for 16-26 weeks. 7,12-DMBA was almost two orders of magnitude more active as a tumor-initiator than 7- and 12-methylbenz[a]anthracene. Methyl substitution at the 7- and 7,12-positions of benz[a]anthracene was significantly more effective in the enhancement of tumorigenic activity than fluorine substitution at these positions. Although 7-fluorobenz[a]anthracene, 12-fluorobenz[a]anthracene, and 7,12-difluorobenz[a]anthracene had only 0.15, 0.26, and less than 0.005 times the tumor-initiating activity of their respective methyl-substituted derivatives, they were severalfold more active than benz[a]anthracene. 7-Fluorobenz[a]anthracene was slightly less active than 12-fluorobenz[a]anthracene, whereas 7-methylbenz[a]anthracene was about twofold more than 12-methylbenz[a]anthracene. For 7,12-di-substituted benz[a]anthracenes, 7-methyl-12-fluorobenz[a]anthracene was more than twice as tumorigenic as 7-fluoro-12-methylbenz[a]anthracene, but each was individually more active than 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene, respectively. Both fluorinated compounds were much less active than 7,12-DMBA. Substitution of fluorine or methyl at the 5-position of 7-methylbenz[a]anthracene and substitution of fluorine at the 5-position of 12-methylbenz[a]anthracene dramatically reduced their tumorigenic activity.

Fluorine substitution as a probe for the role of the 6-position of benzo[a]pyrene in carcinogenesis.

The tumorigenic activities of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-F-BP) were compared to determine whether an unsubstituted 6-position is important for the carcinogenic effect of BP. Highly purified samples of 6-F-BP and BP had similar activities for the induction of lung adenomas in Swiss Webster mice treated before weaning. The 6-fluoro derivative, however, had about one-half as much activity as BP for the initiation of skin papillomas in CD-1 mice. Similarly, 6-F-BP (approximately equal to 90% purity) had about one-half the activity of BP for the induction of skin tumors in C57BL/6J mice given repetitive treatments of the hydrocarbons and for the induction of sarcomas in C3H/fCum mice given a single sc injection. 6-F-BP (approximately equal to 90% purity) had activity similar to that of BP for induction of sarcomas at the sc injection site in Fischer 344 rats. These results and related data indicate the need for detailed metabolic studies whenever fluorine substitution is used as a probe to assess the role of the unsubstituted position in the carcinogenicity of the parent compound.

Bacterial and mammalian cell mutagenicity of four optically active bay-region 3,4-diol-1,2-epoxides and other derivatives of the nitrogen heterocycle dibenz[c,h]acridine.

The mutagenic activities of the enantiomers of the diastereomeric pair of bay-region 3,4-diol-1,2-epoxides of the nitrogen heterocycle, dibenz[c,h]acridine, have been evaluated in histidine-dependent strains of Salmonella typhimurium and in an 8-azaguanine-sensitive line of Chinese hamster cells. In strains TA 98 and TA 100 of S. typhimurium the pair of enantiomers with [1R,2S,3S,4R] and [1S,2R,3R,4S] absolute configuration and the benzylic hydroxyl group trans to the epoxide oxygen are 2 to 4 times more mutagenic than the [1S,2R,3S,4R] and [1R,2S,3R,4S] isomers in which the benzylic hydroxyl and epoxide oxygen are cis. In both strains of bacteria there is very little difference in mutagenic activity between the enantiomers of each diastereomer. In contrast to these results in bacteria, the bay-region 3,4-diol-1,2-epoxide isomer with [1R,2S,3S,4R] absolute configuration is 5 to 7 times more mutagenic to Chinese hamster V79 cells than are the other 3 isomers. The enantiomeric pair of bay-region tetrahydro-1,2-epoxides of dibenz[c,h]acridine are at least 7 times more mutagenic than the diol-epoxides in the Salmonella assay, and no difference in mutagenic activity is observed between enantiomers. In the Chinese hamster V79 cell system, however, the tetrahydro-1,2-epoxide with [1R,2S] absolute configuration is 2- to 3-fold more mutagenic than its enantiomer with [1S,2R] absolute configuration. Homogeneous rat liver epoxide hydrolase does not catalyze the hydration of the diol-epoxide isomers to nonmutagenic products, although the tetrahydroepoxides, especially the tetrahydro-3,4-epoxide, are metabolized by the enzyme. Results of metabolic activation experiments with the bacterial mutagenesis system and microsomes from Aroclor 1254-treated rats are consistent with the mutagenicity data described above and support the concept that dibenz[c,h]acridine is metabolically activated to a bay-region diol-epoxide. Notably: (a) 3,4-dihydrodibenz[c,h]acridine, the expected precursor of a bay-region tetrahydroepoxide, is metabolized to a potent mutagen; (b) racemic dibenz[c,h]acridine 3,4-dihydrodiol is metabolized to products which are several-fold more mutagenic than are products of the metabolism of dibenz[c,h]acridine or its 1,2- or 5,6-dihydrodiols; and (c) the tetrahydro-3,4-diol, which lacks the isolated bay-region double bond, is not metabolically activated to a bacterial mutagen.

Tumorigenicity of optical isomers of the diastereomeric bay-region 3,4-diol-1,2-epoxides of benzo(c)phenanthrene in murine tumor models.

Tumorigenic activities of the (+)- and (-)-enantiomers of the diastereomeric, bay-region benzo(c)phenanthrene 3,4-diol-1,2-epoxides were evaluated in two mouse tumor models. In an initiation-promotion experiment on mouse skin, a single topical application of 10, 25, or 75 nmol of the compounds was followed by 20 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate. Of the four optical isomers of the bay-region diol epoxides, (-)-(R,2S,3S,4R)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrogenzo(c )phenanthrene [(-)-diol epoxide-2] and (+)-(1R,2S,3R,4S)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo(c) -phenanthrene [(+)-diol epoxide-1] had equally high tumor-initiating activity while (+)-[1S,2R,3R,4S]-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo (c)phenanthrene [(+)-diol epoxide-2] had less than one-half of the activity of (-)-diol epoxide-2 and (+)-diol epoxide-1. (-)-(1S,2R,3S,4R)-3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo(c) -phenanthrene [(-)-diol epoxide-1] was inactive at the doses tested. In newborn mice, (-)-diol epoxide-2 was almost 10-fold more active in producing lung tumors (average number of lung tumors/mouse) than the next most active compound, (+)-diol epoxide-2, at a total dose of 10 nmol. The enantiomers of diol epoxide-1 were inactive at this dose. When the total dose of each optical isomer was increased to 50 nmol, (-)-diol epoxide-1 was still inactive, and (+)-diol epoxide-1 produced a significant number of lung tumors (0.9 lung tumor/mouse), but this isomer still had less than 10% of the activity of the (+)- and (-)-diol epoxide-2 isomers. (-)-Diol epoxide-2, but none of the other optical isomers, also produced a significant incidence of hepatic tumors at the higher dose, and this compound was found to be the most tumorigenic bay-region diol epoxide ever tested in newborn mice. Racemic diol epoxide-1 had approximately 1% of the tumorigenic activity of racemic diol epoxide-2 in newborn mice, but both racemates had equal tumor-initiating activity on mouse skin. These results dramatically illustrate the complexities involved in ranking the relative tumorigenic activities of compounds in different tumor models.

High tumorigenicity of the 3,4-dihydrodiol of 7-methylbenz[c]acridine on mouse skin and in newborn mice.

The tumorigenicities of 7-methylbenz[c]acridine (7MB[c]ACR) and its five metabolically possible trans-dihydrodiols were determined in two mouse tumor models. In initiation-promotion studies on mouse skin, a single topical application of 0.15 to 0.75 mumol of compound was followed 9 days later by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate for 20 wk. Comparison of the average number of skin tumors per mouse indicated that 7MB[c]ACR 3,4-dihydrodiol, the metabolic precursor of a bay-region diol-epoxide, was 4- to 6-fold more active than the parent compound as a tumor initiator. The 1,2-, 5,6-, 8,9-, and 10,11-dihydrodiols of 7MB[c]ACR had no significant tumor-initiating activity at the doses tested. In newborn mice, a total dose of 0.35 mumol of compound was administered i.p. during the first 15 days of life, and tumorigenic activity was determined when the mice were 32 to 36 wk old. 7MB[c]ACR 3,4-dihydrodiol induced about 8-fold more pulmonary tumors per mouse and 9-fold more hepatic tumors per male mouse than the parent aza-substituted hydrocarbon. The other four dihydrodiols of 7MB[c]ACR had no significant tumorigenic activity. The high tumorigenic activity of 7MB[c]ACR 3,4-dihydrodiol in both tumor models suggests that a bay-region 3,4-diol-1,2-epoxide may be an ultimate carcinogenic metabolite of 7MB[c]ACR. 7MB[c]ACR was at least 5-fold more active as a tumor initiator on mouse skin than was the unsubstituted aza-aromatic compound, benz[c]acridine. This latter result indicates that substitution of a methyl group at position 7 of benz[c]acridine leads to enhanced tumor-initiating activity, as has been previously demonstrated for benz[a]anthracene and its 7-methyl derivative.

Dose-dependent differences in the profile of mutations induced by (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in Chinese hamster V-79 cells.

Chinese hamster V-79 cells were exposed to a high dose (0.30-0.48 microM; 32% cell survival), an intermediate dose (0.04-0.10 microM; 100% cell survival) or a low dose (0.01-0.02 microM; 97% cell survival) of (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene [(+)-BPDE] which is the ultimate carcinogenic metabolite of benzo(a)pyrene. The mutation frequency for cells treated with dimethyl sulfoxide vehicle or with low, intermediate or high dose of (+)-BPDE were 1, 10, 52 or 514 8-azaguanine-resistant colonies/10(5) survivors, respectively. Independent 8-azaguanine-resistant clones were isolated, and complementary DNAs were prepared by reverse transcription. The coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene was amplified by the polymerase chain reaction and sequenced. Altogether, 368 (+)-BPDE-induced mutant clones were examined. At all doses, base substitutions were the most prevalent mutations observed (about 72% of the mutant clones), followed by exon deletions (about 26% of the mutant clones) and frame-shift mutations (about 6% of the mutant clones). At the high cytotoxic dose, 7 of 120 base substitutions occurred at AT base pairs (6%) and 113 at GC base pairs (94%). At the intermediate noncytotoxic dose, 20 of 82 base substitutions occurred at AT base pairs (24%) and 62 at GC base pairs (76%). At the low noncytotoxic dose, 27 of 76 base substitutions were at AT base pairs (36%) and 49 were at GC base pairs (64%). The results indicated that decreasing the dose of (+)-BPDE decreased the proportion of mutations at GC base pairs and increased the proportion of mutations at AT base pairs. At the dose of (+)-BPDE was decreased, there was a dose-dependent decrease in the proportion of GC-->TA transversions (from 69% to 42% of the base substitutions) and a dose-dependent increase in the proportion of AT-->CG transversions (from 1% to 25% of the base substitutions). The data also indicated dose-dependent differences in (+)-BPDE-induced exon deletions and hot spots for base substitutions at GC and AT base pairs. Although more than 99% of the (+)-BPDE-induced mutations at guanine occurred on the nontranscribed strand of DNA, (+)-BPDE-induced mutations at adenine occurred on both the transcribed and nontranscribed strands. The ratio of mutations at adenine on the transcribed strand to mutations at adenine on the nontranscribed strand was 35:19 in (+)-BPDE-treated V-79 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Mutagenicity of dihydrodiols and diol epoxides of dibenz[a, h]acridine in bacterial and mammalian cells.

Bay-region diol epoxides are ultimate carcinogenic metabolites of a number of polycyclic aromatic compounds. Dibenz[a, h]acridine can form two diastereomeric pairs of these diol epoxides which are not positionally equivalent as a result of the nitrogen atom at position 7. We have assessed the structure-activity relationships resulting from heterocyclic nitrogen substitution by examining the mutagenic activity of these four bay-region diol epoxides of dibenz[a,h]acridine in both bacterial and mammalian cells. In strains TA98 and TA100 of Salmonella typhimurium, the diastereomeric 10,11-diol-8,9-epoxides were 20 to 40 times more mutagenic than the corresponding 3,4-diol-1,2-epoxides. Furthermore, in strain TA100, dibenz[a,h]acridine 10,11-dihydrodiol, the expected metabolic precursor of the 10,11-diol-8,9-epoxide, was metabolically activated by rat hepatic microsomes up to a 12-fold greater extent than the 3,4-dihydrodiol. In Chinese hamster V79 cells, the 10,11-diol-8,9-epoxide diastereomers were 20 to 80 times more mutagenic than their 3,4-diol-1,2-epoxide counterparts. Quantum mechanical calculations of the predicted ease of benzylic carbocation formation at C-1 and C-8 from the diol epoxides indicate that the 3,4-diol-1,2-epoxides should be less reactive due to resonance destabilization of the C-1 carbocation as a result of the electronegative nitrogen atom. Decreased chemical reactivity of 3,4-diol-1,2-epoxides may explain their decreased mutagenic activity.

Mutagenicity of the enantiomers of the diastereomeric bay-region benz(a)anthracene 3,4-diol-1,2-epoxides in bacterial and mammalian cells.

Enantiomers of the diastereomeric pair of bay-region benz(a)anthracene 3,4-diol-1,2-epoxides in which the benzylic 4-hydroxyl group and epoxide oxygen are either cis (isomer 1) or trans (isomer 2) were evaluated for mutagenic activity in two histidine-dependent strains of Salmonella typhimurium, as well as in an 8-azaguanine-sensitive Chinese hamster cell line. In strain TA 98 of S. typhimurium, the diol-epoxide with (1S,2R,3R,4S) absolute configuration [(-)-diol-epoxide 2] was the most active isomer, although there was less than a 3-fold difference in the mutagenicity of the four diol-epoxides. However, in strain TA 100 of S. typhimurium, the enantiomeric diol-epoxide with (1R,2S,3S,4R) absolute configuration [(+)-diol-epoxide 2] was the most active diol-epoxide, and the two isomers with (3S,4R) absolute configuration [(-)-diol-epoxide 1 and (+)-diol-epoxide 2] were three to eight times more active than were the two isomers with (3R,4S) configuration. The highest degree of sensitivity to absolute configuration was observed in Chinese hamster V79 cells, in which the (1R,2S,3S,4R) isomer [(+)-diol-epoxide 2] was from three to 20 times more mutagenic than were the other three isomers. This metabolically predominant (+)-diol-epoxide 2 isomer, which has high activity in strain TA 100 of S. typhimurium and the Chinese hamster V79 cells, has the same absolute configuration as do the bay-region diol-epoxide isomers of benzo(a)pyrene and chrysene that have been shown previously to be exceptionally mutagenic to mammalian cells and highly tumorigenic in mice. Analysis of the mutagenic activity of the (+)- and (-)-isomers of the 1,2- and 3,4-tetrahydroepoxides of benz(a)anthracene revealed only small enantiomeric differences in strain TA 98 of S. typhimurium (2.5 fold) and little, if any, differences (less than 1.5-fold) in the other two mutagenicity systems. However, the extent to which the four tetrahydroepoxides were converted to nonmutagenic products by homogeneous microsomal epoxide hydrolase (EC 3.3.2.3) indicated marked differences in the stereoselectivity of the enzyme. (-)-(3R,4S)-Epoxy-1,2,3,4-tetrahydrobenz(a)anthracene appears to be an exceptionally good substrate for epoxide hydrolase.

Tumor-initiating activity of benz[c]acridine and twelve of its derivatives on mouse skin.

Benz[c]acridine (B[c]ACR) and 12 of its derivatives, including the 5 metabolically possible trans-dihydrodiols, the diastereomeric bay-region diol-epoxides, 2 non-bay-region diol-epoxides, and the K-region arene oxide, were tested for tumor-initiating activity on mouse skin. A single topical application of 0.4 to 2.5 mumol of compound was followed 12 days later by twice-weekly applications of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate for 25 weeks. B[c]ACR was a weak tumor initiator on mouse skin, producing a 37% tumor incidence and 1.33 tumors/mouse at the 2.5-mumol dose. Of the five metabolically possible trans-dihydrodiols of B[c]ACR, only trans-3,4-dihydroxy-3,4-dihydro-B[c]ACR had significant tumor-initiating activity. This compound was at least 6-fold more active than was the parent compound at the three doses tested. The diastereomeric bay-region diol-epoxides, in which the epoxide oxygen is either cis(isomer 1) or trans (isomer 2) to the benzylic hydroxyl group, each had significant tumor-initiating activity, although isomer 2 was at least 5-fold more active than was isomer 1 and had activity equal to that of its potential metabolic precursor, trans-3,4-dihydroxy-3,4-dihydro-B[c]ACR. Two non-bay-region diol-epoxides (isomer 2 of the 8,9-diol-10,11-epoxide and the 10,11-diol-8,9-epoxide) and the 5,6-arene oxide (K-region) were inactive at the doses tested. 3,4-Dihydro-B[c]ACR, the potential metabolic precursor of a bay-region tetrahydroepoxide, was the most potent tumor initiator analyzed in the present study. At an initiating dose of 0.4 mumol, this compound produced a 97% tumor incidence and 7.90 tumors/mouse after 15 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate. These results suggest that B[c]ACR, the N-12 analogue of benz[a]anthracene, undergoes metabolic activation to an ultimate carcinogenic metabolite via formation of a bay-region diol-epoxide, as has already been demonstrated for benz[a]anthracene.

Carcinogenicity of 2-hydroxybenzo(a)pyrene and 6-hydroxybenzo(a)pyrene in newborn mice.

Benzo(a)pyrene (BP), 2-hydroxybenzo(a)pyrene (2-HOBP), and 6-hydroxybenzo(a)pyrene (6-HOBP) were tested for tumorigenicity by i.p. injection into newborn mice. The mice were treated sequentially with 200, 400, and 800 nmol of compound on the first, eighth and fifteenth day of life, and the animals were killed at 24 weeks of age. Treatment with 2-HOBP caused about 4-fold more pulmonary tumors than BP, while 6-HOBP had little or no tumorigenic activity. Newborn mice treated with 2-HOBP, BP, and 6-HOBP had a 98, 81, and 11% incidence of pulmonary adenomas with an average of 24, 6.4, and 0.11 adenomas per mouse, respectively. In the control group, 7.5% of the animals had pulmonary adenomas with an average of 0.08 adenoma per mouse. When 25, 50, or 100 nmol of BP or 2-HOBP was applied to mouse skin once every 2 weeks for 60 weeks, both compounds had about the same carcinogenic activity. These results demonstrate the importance of evaluating the carcinogenic potential of chemicals in more than one tumor system. BP and 2-HOBP were tested for mutagenicity towards two strains of Salmonella typhimurium and towards Chinese hamster V79 cells in the presence of hepatic microsomes from rats pretreated with Aroclor 1254. The products formed during the metabolism of 2-HOBP or BP by liver microsomes had significant mutagenic activity.

Marked differences in the tumor-initiating activity of optically pure (+)- and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene on mouse skin.

The ability of optically pure (+)- and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene to initiate skin tumors in mice was determined with a two-stage tumorigenesis system. A single application of 50 to 200 nmoles of (+)- or (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene to the backs of CD-1 mice followed by twice-weekly applications of 12-O-tetradecanoyl-phorbol-13-acetate revealed that the (-)-enantiomer was 5- to 10-fold more potent than was the (+)-enantiomer as a tumor initiator at the three dosage levels tested. When the tumor-initiating activities of the (+)0 and (-)-enantiomers of trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene were compared to the activity of benzo(a)pyrene at an equimolar dose, the (-)-enantiomer was more active while the (+)-enantiomer was considerably less active. This is the first report of differences in the carcinogenic activity between optical enantiomers.

Mutagenicity and cytotoxicity of benzo(a)pyrene arene oxides, phenols, quinones, and dihydrodiols in bacterial and mammalian cells.

Twenty-nine benzo(a)pyrene derivatives were tested for mutagenic acitivity without metabolic activation in Salmonella typhimurium strains TA98, TA100, and TA1538 and in Chinese hamster V79 cells. The compounds studied included 4 arene oxides, all 12 isomeric phenols, 5 quinones, and 8 dihydrodiols. Benzo(a)pyrene 4,5-oxide was the most mutagenic of the compounds tested in both the bacterial and mammalian systems. The other arene oxides [benzo(a)pyrene 7,8-, 9,10-, and 11,12-oxides] were only weakly mutagenic in the S. typhimurium strains. However, in Chinese hamster V79 cells benzo(a)pyrene 11,12-oxide. Among the phenols, 6-hydroxybenzo(a)pyrene and 12-hydroxybenzo(a)pyrene were moderately mutagenic in strain TA98 of S. typhimurium, and 6-hydroxybenzo(a)pyrene was moderately mutagenic in V79 cells. The other 10 phenols, 5 quinones [benzo(a)pyrene 1,6-, 3,6-, 4,5-, 6, 12-, and 11,12-quinones] and 8 dihydrodiols [benzo(a)pyrene cis-4,5,trans-4,5-, cis-7,8-, trans-7,8-, cis-9,10-, trans-9,10-, cis-11,12-, and trans-11, 12-dihydrodiols] were eitherinactive or only weekly mutagenic. 1-Hydroxybenzo(a)pyrene and 3-hydroxybenzo(a)pyrene were weakly mutagenic in strain TA98 of S. typhimurium, and benzo(a)pyrene 7,8-dihydrodiol was weakly mutagenic in V79 cells. Benzo(a)pyrene 11,12-quinone was extremely cytotoxic to the V79 cells but had no observable toxicity in the bacterial strains.

Mutagenicity and cytotoxicity of benzo(a)pyrene benzo-ring epoxides.

Four benzo-ring epoxides of the environmental carcinogen benzo(a)pyrene (BP) were tested for mutagenic and cytotoxic activity in 3 strains of Salmonella typhimurium (TA1538, TA98, and TA100) and in Chinese hamster V79 cells. Although very unstable in aqueous solution, 7beta,8alpha-dihydroxy-0beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (diol epoxide 1), with the 7-hydroxyl group on the same face of the molecule as the epoxide oxygen, was 1.5 to 4 times as mutagenic in the bacterial strains as was its more stable stereoisomer 7beta,8alpha-dihydroxy-9alpha,10beta-epoxy-7,8,9.10-tetrahydrobenzo(a)pyrene (diol epoxide 2). In V79 cells, diol epoxide 1 had one-third the mutagenic activity of diol epoxide 2 but was at least 10 times more labile than diol epoxide 2 in the tissue culture medium. The half-life of diol epoxide 1 in tissue culture medium was about 30 sec, whereas the half-life of diol epoxide 2 was between 6 and 12 min. 9,10-Epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, which is saturated in the benzo ring, is also very unstable and has mutagenic activity equal to or greater than diol epoxide 1 in the bacterial and mammalian cells. 7,8-Epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was more stable in aqueous solution than any of the 9,10-epoxides of BP but was much less mutagenic in both the bacterial and mammalian cells. In v79 cells, diol epoxides 1 and 2 and 9,10-opoxy-7,8,9,10-tetrahydrobenzo(a)pyrene were more than 40 times more cytotoxic than 7,8-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. The mutagenicity of the 2 tetrahydro epoxides toward strain TA98 of S. typhimurium was readily abolished by purified epoxide hydrase, whereas the mutagenic activity of the 2 diol epoxides was relatively unaffected by coincubation with the enzyme.

High stereoselectivity among the optical isomers of the diastereomeric bay-region diol-epoxides of benz(a)anthracene in the expression of tumorigenic activity in murine tumor models.

The tumorigenicity of the (+)- and (-)-enantiomers of the diastereomeric bay-region benz(a)anthracene 3,4-diol-1,2-epoxides was evaluated in two mouse tumor models. In an initiation-promotion experiment on mouse skin, a single topical application of 0.1 or 0.4 mumol of the benz(a)anthracene diol-epoxides was followed by 25 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate. Of the four isomers of the bay-region diol-epoxides, only (+)-[1R,2S,3S,4R]-3,4-dihydroxy-1,2-epoxy-1,2,3,4- tetrahydrobenz(a)anthracene [(+)-diol-epoxide-2] and (+)-[1R,2S,3S,4S]-3,4-dihydroxy-1,2-epoxy-1,2,3,4- tetrahydrobenz(a)anthracene [(+)-diol-epoxide-1] had significant tumor-initiating activity. (+)-Diol-epoxide-2 was approximately 4-fold more active as a tumor initiator on mouse skin than was (+)-diolepoxide-1 at both doses tested. In newborn mice, a total of 0.14 mumol of compound, divided into three doses, was administered i.p. on the first, eighth, and fifteenth day of life, and tumorigenic activity was determined when the mice were 26 to 32 weeks of age. As was observed in the initiation-promotion experiment on mouse skin, only two of the four optical isomers of the bay-region diol-epoxides produced a significant tumor incidence. (+)-Diol-epoxide-2 induced a 100% incidence of lung tumors, with an average of 23.11 tumors/mouse, and was at least 60-fold more active (average number of tumors per mouse) than was (+)-diol-epoxide-1, which produced a 31% lung tumor incidence and 0.38 lung tumors/mouse. (+)-Diol-epoxide-2 was the only optical isomer that induced a significant incidence of hepatic tumors in male mice (31% incidence, 1.17 tumors/mouse). The highly tumorigenic (+)-diol-epoxide-2 isomer with [R,S,S,R] absolute configuration has the same absolute configuration as does the highly tumorigenic isomer of the bay-region diol-epoxides of benzo(a)pyrene and chrysene.

Mutagenicity of the enantiomers of the diastereomeric bay-region benzo(c)phenanthrene 3,4-diol-1,2-epoxides in bacterial and mammalian cells.

The mutagenic activities of the enantiomers of the pair of diastereomeric bay-region benzo(c)phenanthrene 3,4-diol-1,2-epoxides were evaluated in histidine-dependent strains of Salmonella typhimurium and in an 8-azaguanine-sensitive Chinese hamster cell line. In strains TA 98 and TA 100 of S. typhimurium, the range in mutagenic activity observed for the four optically active isomers was less than 4- and 2-fold, respectively. The diol-epoxide with (1S,2R,3R,4S) absolute configuration and the benzylic hydroxyl group trans to the epoxide oxygen [(+)-diol epoxide-2] was the most active isomer in both strains. The enantiomeric (-)-diol-epoxide-2 isomer, with (1R,2S,3S,4R) absolute configuration identical to that of the exceptionally tumorigenic (+)-diol-epoxide-2 isomers of benzo(a)pyrene, benz(a)anthracene, and chrysene, was the least active isomer in strain TA 98 (27%) and the second most active isomer in strain TA 100 (90%). In Chinese hamster V79 cells (-)-diol-epoxide-2 was the most active of the four benzo(c)phenanthrene isomers, and a 4- to 5-fold range in mutagenic activity was observed. The differences in mutagenic activity between the four bay-region diol-epoxide isomers of benzo(c)phenanthrene in the three test systems are relatively small when compared with results from similar studies with optically active bay-region diol-epoxide isomers of three other polycyclic aromatic hydrocarbons, and may be explicable, in part, by a tendency of the hydroxyl groups of benzo(c)phenanthrene diol-epoxides to adopt comparable pseudodiequatorial conformations.

Tumorigenicity of dihydrodiols and diol-epoxides of benz[c]acridine in newborn mice.

The tumorigenicity of benz[c]acridine (B[c]ACR) and a number of its derivatives, including the five metabolically possible transdihydrodiols, the diastereomeric bay-region diol-epoxides, two non-bay-region diol-epoxides, and the K-region 5,6-oxide, were assessed in newborn mice. A total dose of 0.50 or 1.05 mumol of compound was administered i.p. to preweanling mice, and tumorigenic activity was determined when the mice were 33 to 37 weeks old. B[c]ACR was a weak carcinogen producing an average of 2.5 lung tumors/mouse and 0.15 liver tumor/male mouse at the 1.05-mumol dose. Of the five metabolically possible trans-dihydrodiols of B[c]ACR, only trans-3,4-dihydroxy-3,4-dihydro-B[c] ACR (B[c]ACR (B[c]ACR 3,4-dihydrodiol) had high tumorigenic activity. B[c]ACR 3,4-dihydrodiol induced 2- and 10-fold more pulmonary and hepatic tumors, respectively, than did the parent compound while the trans-1,2-, 5,6-, 8,9-, and 10,11-dihydrodiols had very little or no tumorigenic activity. Both of the diastereomeric bay-region 3,4-diol-1,2-epoxides, in which the epoxide oxygen is either cis (isomer 1) or trans (isomer 2) to the benzylic hydroxyl group, had tumorigenic activity. Isomer 2 was the most tumorigenic derivative tested, inducing at least 60, 7, and 12 times more lung tumors per mouse than did isomer 1, B[c]ACR 3,4-dihydrodiol and B[c]ACR, respectively. The K-region 5,6-oxide and two non-bay-region diol-epoxides (isomer 2 of B[c]ACR 8,9-diol-10,11-epoxide and B[c]ACR 10,11-diol-8,9-epoxide) were weakly active or inactive at the dose tested. The demonstration that B[c]ACR 3,4-diol-1,2-epoxide-2 is exceptionally tumorigenic and that its metabolic precursor, B[c]ACR 3,4-dihydrodiol, is more active than the parent hydrocarbon, B[c]ACR, support the concept that isomer 2 of the bay-region diol-epoxide may be an ultimate carcinogenic metabolite of B[c]ACR.