SH2B3 aberrations enriched in iAMP21 B lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) represents the most common childhood malignancy. Although survival for pediatric B-ALL has approached 90%, variability in outcome among and within cytogenetically defined subgroups persists. While G-banding and fluorescence in situ hybridization (FISH) have been used to characterize leukemic clones, there is added value of chromosomal microarray and next generation sequencing in screening genome-wide for copy number aberrations, copy neutral loss of heterozygosity and nucleotide variations. Evaluation of novel genetic aberrations can provide information about the biologic mechanisms of cytogenetically defined subgroups associated with poor prognosis, explain heterogeneity in patient outcome and identify novel targets for therapeutic intervention. The high risk B-ALL intrachromosomal amplification of chromosome 21, (iAMP21), subtype is characterized by amplification of a region of chromosome 21 that typically encompasses the RUNX1 gene and is associated with poor prognosis. Analysis of chromosomal microarray and FISH data revealed that deletions of SH2B3, encoding a negative regulator of multiple tyrosine kinase and cytokine signaling pathways, are enriched among leukemias harboring iAMP21. Enrichment of SH2B3 aberrations in the iAMP21 subtype may indicate that loss of SH2B3 contributes to disease progression and raises the possibility that these leukemias may be sensitive to tyrosine kinase inhibitors.

Further Evidence of Contrasting Phenotypes Caused by Reciprocal Deletions and Duplications: Duplication of NSD1 Causes Growth Retardation and Microcephaly.

Microduplications of the Sotos syndrome region containing NSD1 on 5q35 have recently been proposed to cause a syndrome of microcephaly, short stature and developmental delay. To further characterize this emerging syndrome, we report the clinical details of 12 individuals from 8 families found to have interstitial duplications involving NSD1, ranging in size from 370 kb to 3.7 Mb. All individuals are microcephalic, and height and childhood weight range from below average to severely restricted. Mild-to-moderate learning disabilities and/or developmental delay are present in all individuals, including carrier family members of probands; dysmorphic features and digital anomalies are present in a majority. Craniosynostosis is present in the individual with the largest duplication, though the duplication does not include MSX2, mutations of which can cause craniosynostosis, on 5q35.2. A comparison of the smallest duplication in our cohort that includes the entire NSD1 gene to the individual with the largest duplication that only partially overlaps NSD1 suggests that whole-gene duplication of NSD1 in and of itself may be sufficient to cause the abnormal growth parameters seen in these patients. NSD1 duplications may therefore be added to a growing list of copy number variations for which deletion and duplication of specific genes have contrasting effects on body development.

Reduced-intensity conditioning is effective and safe for transplantation of patients with Shwachman-Diamond syndrome.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only potentially curative treatment for the BM dysfunction seen in patients with Shwachman-Diamond syndrome (SDS). Historically, these patients have fared poorly with intensive conditioning regimens with increased regimen-related toxicity especially involving the heart and lungs. We report our institutional experience with a reduced-intensity-conditioning protocol in seven patients with SDS and BM aplasia or myelodysplastic syndrome/AML. The preparative regimen consisted of Campath-1H, fludarabine and melphalan. Four patients received matched related marrow and three received unrelated stem cells (two PBSCs and one marrow). All but one was 8 of 8 allele HLA matched. All patients established 100% donor-derived hematopoiesis. No patient in this cohort developed grades III-IV GVHD. One patient had grade II skin GVHD that responded to systemic corticosteroids and one had grade I skin GVHD, treated with topical corticosteroids. Two out of seven patients developed bacterial infections in the early post transplant period. Viral infections were seen in four out of seven patients and were successfully treated with appropriate antiviral therapy. All patients are currently alive. These data indicate that HSCT with reduced-intensity conditioning is feasible in patients with SDS and associated with excellent donor cell engraftment and modest morbidity.

Single- and multiple-dose pharmacokinetics of ziprasidone in healthy young and elderly volunteers.

AIMS: To compare the pharmacokinetics of ziprasidone in healthy young (18-45 years) men and women, and healthy elderly (> or = 65 years) men and women. METHODS: Eight young men, 11 young women, 8 elderly men and 8 elderly women were given oral ziprasidone 40 mg day(-1), in two evenly divided daily doses, for 7 days, followed by a single 20 mg dose on day 8. Serum samples were collected immediately before the morning dose on days 1-8, for up to 12 h after dosing on day 1 and for up to 96 h after dosing on day 8. The resulting data were used to derive pharmacokinetic parameters of ziprasidone in each age and gender group. RESULTS: Steady-state serum concentrations of ziprasidone were achieved within 2-3 days. The steady-state pharmacokinetics of ziprasidone, determined 8 days after the initiation of treatment, were similar in the young men, elderly men and young women. Assessment of gender effects by analysis of variance revealed statistically significant differences in Cmax (85 vs. 69 ng ml(-1) and tmax (3.19 vs. 4.81 h) but no differences in AUC(0,12 h) or lambda(z). Assessment of age effects by analysis of variance revealed statistically significant differences in AUC(0,12 h) (560 vs. 465 ng ml(-1) h), Cmax (85 vs. 69 ng ml(-1) and lambda(z) (0.126 vs. 0.197 l h(-1) but no difference in tmax. Assessment of age and gender effects by analysis of covariance, with body weight as the covariate, did not reveal any significant differences. The mean t(1/2), z in the young men, young women, elderly men and elderly women were 3.1, 4.1, 5.7 and 5.3 h, respectively. Standard deviations of the means for the pharmacokinetic parameters for the elderly women tended to be large. CONCLUSIONS: The influence of age and gender on the pharmacokinetics of ziprasidone is not clinically significant.

The pharmacokinetics of ziprasidone in subjects with normal and impaired hepatic function.

AIMS: To assess whether hepatic impairment influences the pharmacokinetics of ziprasidone. METHODS: Thirty subjects with normal hepatic function or a primary diagnosis of clinically significant cirrhosis (Child-Pugh A or B) were enrolled into an open-label, multicentre, multiple-dose study. The subjects with chronic, stable hepatic impairment and the matched control subjects received ziprasidone 40 mg day(-1), given orally with food, as two divided daily doses for 4 days and a single 20 mg dose on the morning of day 5. Pharmacokinetic variables were determined from multiple venous blood samples collected on days 1 and 5. Liver function was evaluated quantitatively using antipyrine. RESULTS: On day 1 there were no statistically significant differences in the pharmacokinetics (AUC(0,12 h), Cmax, tmax) of ziprasidone between the two groups. On day 5 there were no statistically significant differences in the Cmax or tmax for ziprasidone between the two groups. The mean AUC(0,12 h) for ziprasidone was statistically significantly greater in the hepatically impaired subjects compared with the normal subjects (590 ng ml(-1) h vs. 467 ng ml(-1) h, P = 0. 042). However, the AUC(0,12 h) increased by only 26% in the cirrhotic group compared with the matched control group. The ziprasidone lambda(z) in the subjects with normal hepatic function was statistically significantly greater than that in the hepatically impaired subjects (P<0.001). There was no correlation between antipyrine lambda(z) and ziprasidone lambda(z) in the subjects with normal hepatic function or in those with hepatic impairment. CONCLUSIONS: The findings of this study indicate that mild to moderate hepatic impairment does not result in clinically significant alteration of ziprasidone pharmacokinetics.

The pharmacokinetics of ziprasidone in subjects with normal and impaired renal function.

AIMS: To assess whether renal impairment influences the pharmacokinetics of ziprasidone, and to determine whether ziprasidone is cleared via haemodialysis. METHODS: Thirty-nine subjects with varying degrees of renal impairment were enrolled into an open-label, multicentre, multiple-dose study and assigned to four groups according to their renal function: normal (group 1, creatinine clearance > 70 ml min(-1); mildly impaired (group 2, creatinine clearance 30-60 ml min(-1); moderately impaired (group 3, creatinine clearance 10-29 ml min(-1), and severely impaired (group 4, requiring haemodialysis three times-a-week). Subjects received ziprasidone 40 mg day(-1), given orally with food, as two divided daily doses for 7 days and a single 20 mg dose on the morning of day 8. Pharmacokinetic variables were determined from multiple venous blood samples collected on days 1 and 8 (haemodialysis day for subjects with severe renal impairment). Additional samples were collected from subjects with severe renal impairment on day 7 (nonhaemodialysis day). RESULTS: On day 1 there were no statistically significant differences in the pharmacokinetics (AUC(0, 12 h), Cmax, tmax) of ziprasidone among subjects with normal renal function and those with mild, moderate and severe renal impairment. The AUC(0,12 h) and Cmax in subjects with mildly impaired renal function were statistically significantly greater than in those with moderately impaired renal function (P = 0.0163-0.0385). The mean AUC(0,12 h) was 272, 370, 250 and 297 ng ml(-1) h in groups 1, 2, 3 and 4, respectively. Corresponding mean Cmax values were 47, 61, 41 and 50 ng ml(-1) and corresponding mean tmax values were 5, 6, 5 and 5 h. On day 8 there were no statistically significant differences in the pharmacokinetics (AUC(0,12 h), Cmax, tmax, lambda(z), Fb) of ziprasidone among subjects with normal renal function and those with moderate or severe renal impairment. The AUC(0,12 h) in subjects with mild renal impairment was statistically significantly greater than those in the other three groups (P = 0.0025-0.0221), but this was not considered clinically significant. The mean AUC(0,12 h) were 446, 650, 389 and 427 ng ml(-1) h in groups 1, 2, 3 and 4, respectively. Corresponding mean Cmax values were 68, 93, 54 and 70 ng ml(-1), corresponding mean tmax values were 4, 5, 4 and 5 h and corresponding mean lambda(z) were 0.14, 0.11, 0.14 and 0.17 h(-1). The mean percentage Fb was 99.84-99.88% across all groups and the mean t(1/2),z ranged from 4.2 to 6.4 h. Comparison of the mean AUC(0,12 h) and Cmax values in subjects with severe renal impairment on day 7 with those on day 8 suggested that haemodialysis does not have a clinically significant effect on the pharmacokinetics of ziprasidone. CONCLUSIONS: The findings of this study indicate that mild-to-moderate impairment of renal function does not result in clinically significant alteration of ziprasidone pharmacokinetics and therefore does not necessitate dose adjustment.

Mutational analysis of the tuberous sclerosis gene TSC2 in patients with pulmonary lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare disorder limited almost exclusively to women of reproductive age. LAM affects about 5% of women with tuberous sclerosis complex (TSC). LAM also occurs in women who do not have TSC (sporadic LAM). TSC is a tumour suppressor gene syndrome characterised by seizures, mental retardation, and tumours in the brain, heart, and kidney. Angiomyolipomas, which are benign tumours with smooth muscle, fat, and dysplastic vascular components, are the most common renal tumour in TSC. Renal angiomyolipomas also occur in 63% of sporadic LAM patients. We recently found that 54% of these angiomyolipomas have TSC2 loss of heterozygosity, leading to the hypothesis that sporadic LAM is genetically related to TSC. In this study, we screened DNA from 21 women with sporadic LAM for mutations in all 41 exons of TSC2. Twelve of the patients had known renal angiomyolipomas. No TSC2 mutations were detected. We did find three silent TSC2 polymorphisms. We conclude that patients with sporadic LAM, including those with renal angiomyolipomas, do not have a high frequency of germline mutations in the coding region of TSC2.

Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774: dynamics of receptor inhibition in situ and antitumor effects in athymic mice.

Phosphorylation of tyrosine residues on the epidermal growth factor (EGF) receptor (EGFr) is an important early event in signal transduction, leading to cell replication for major human carcinomas. CP-358,774 is a potent and selective inhibitor of the EGFr tyrosine kinase and produces selective inhibition of EGF-mediated tumor cell mitogenesis. To assess the pharmacodynamic aspects of EGFr inhibition, we devised an ex vivo enzyme-linked immunosorbent assay for quantification of EGFr-specific tyrosine phosphorylation in human tumor tissue specimens obtained from xenografts growing s.c. in athymic mice. When coupled with pharmacokinetic analyses, this measurement can be used to describe the extent and duration of kinase inhibition in vivo. CP-358,774 is an effective, orally active inhibitor of EGFr-specific tyrosine phosphorylation (ED(50) = 10 mg/kg, single dose). It has a significant duration of action, producing, on average, a 70% reduction in EGFr-associated phosphotyrosine over a 24-h period after a single 100 mg/kg dose. Inhibition of EGFr phosphotyrosine in an ex vivo assay format effectively estimates the potency and degree of inhibition of EGFr-dependent human LICR-LON-HN5 head and neck carcinoma tumor growth. Substantial growth inhibition of human tumor xenografts was achieved with p.o. doses of the compound (ED(50) = 10 mg/kg q.d. for 20 days). Combination chemotherapy with cisplatin produced a significant response above that of cisplatin alone with no detectable effects on body weight or lethal toxicity. Taken together, these observations suggest that CP-358,774 may be useful for the treatment of EGFr-driven human carcinomas.

Cytogenetic analyses of 85 testicular germ cell tumors: comparison of postchemotherapy and untreated tumors.

Cytogenetic analyses of 85 testicular germ cell tumors, of which 54 were karyotypically abnormal, showed recurrent breakpoints at chromosome bands 1p36, 1p13-1qh, 11q23, 19q13, and the pericentromeric regions of the acrocentric chromosomes. Postchemotherapy tumors had significantly more rearrangements of bands 3p25-p26, 6q16-q21, 8p22-p23 when compared with untreated tumors, while untreated tumors had more rearrangements of 9p22-p24 when compared with postchemotherapy tumors. Frequent breakpoints also were identified at 15q15 and 9qh in untreated tumors. Tumors of different histopathology, clinical stage, and treatment status showed no significant differences in the frequencies of i(12p)-positive and i(12p)-negative tumors.

Characterization of multiple 12p rearrangements in testicular germ cell tumor cell line 833K and its subclone 64CP by chromosome microdissection.

Rearrangements of chromosome arm 12p are known to be common in germ cell tumors (GCT). Previous studies, using fluorescence in situ hybridization (FISH) with a whole chromosome 12 painting probe, showed unusual distributions of chromosome 12-derived chromatin in GCT cell line 833K and its cisplatin-resistant subclone, 64CP, located next to AgNOR (silver staining nucleolus organizer regions), some of which were ectopic. In this study, the ectopic stalk regions were shown by FISH to be composed of 18s and 28s rDNA, but were flanked by beta-satellite DNA, which may form a barrier around the rDNA. In order to determine the specific origins of the rearranged chromosome 12 segments, three different derived chromosome 12 regions were isolated from 64CP, using chromosomal microdissection. The microdissected fragments were labeled and hybridized by FISH to normal human chromosomes. All three segments localized to distal 12p; 12p12-->12pter, but with apparently different breakpoints for each segment. Furthermore, three-color FISH experiments with 12p band-specific probes demonstrated that the derivative chromosome 12 regions in 833K also originate from distal 12p (12p12-->p13). These sequences now can be evaluated for degree of overlap or common breakpoints which may be of significance in the development or progression of GCT.

Interphase chromosome painting of paraffin-embedded tissue in the differential diagnosis of possible germ cell tumors.

Germ cell tumors (GCTs) are the most frequent cancer in men aged 15 to 34 years. These tumors are highly responsive to therapy with platinum-containing regimens, and 80% of cases so treated can be considered cured. Cytogenetically, 80% of GCTs have an i(12p) regardless of tumor site or histopathology, and those that are i(12p) negative have other manifestations of 12p amplification. GCTs occasionally arise extragonadally, and such cases can be especially difficult to distinguish from poorly differentiated somatic carcinomas, a situation that poses a diagnostic and treatment dilemma We developed a technique for two-color fluorescence in situ hybridization chromosome painting on nuclei released from paraffin-embedded sections. In four tumors for which GCT was a differential diagnosis, we examined the 12p and 12q chromosome arm distributions by this technique. By use of 12p and 12q painting probes developed by microdissection, 12p and 12q were distinguished and their relative distributions evaluated. In each of the four cases, 12p regions seemed to be rearranged and over-represented relative to 12q regions. In three of the cases, an apparent i(12p) could be identified. These results support a diagnosis of GCT or GCT origin in these four cases. In tumors for which specific cytogenetic abnormalities are known, chromosome painting by fluorescence in situ hybridization using paraffin-embedded tissue is a useful technique to aid in the diagnosis of tumors that are difficult to differentiate. The patients can then be placed on treatment regimens appropriate for their specific tumor type.

Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.

Lymphangiomyomatosis (LAM) is a rare disease, of unknown etiology, affecting women almost exclusively. Lung transplantation is the only consistently effective therapy for LAM. Microscopically, LAM consists of a diffuse proliferation of smooth muscle cells. LAM can occur without evidence of other disease (referred to as "sporadic LAM") or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant tumor suppressor gene syndrome characterized by seizures, mental retardation, and tumors in the brain, heart, skin, and kidney. Renal angiomyolipomas occur in approximately 50% of sporadic LAM patients and in 70% of TSC patients. Loss of heterozygosity (LOH) in the chromosomal region for the TSC2 gene occurs in 60% of TSC-associated angiomyolipomas. Because of the similar pulmonary and renal manifestations of TSC and sporadic LAM, we hypothesized that LAM and TSC have a common genetic basis. We analyzed renal angiomyolipomas, from 13 women with sporadic LAM, for LOH in the regions of the TSC1 (chromosome 9q34) and TSC2 (chromosome 16p13) genes. TSC2 LOH was detected in seven (54%) of the angiomyolipomas. We also found TSC2 LOH in four lymph nodes from a woman with retroperitoneal LAM. No TSC1 LOH was found. Our findings indicate that the TSC2 gene may be involved in the pathogenesis of sporadic LAM. However, genetic transmission of LAM has not been reported. Women with LAM may have low-penetrance germ-line TSC2 mutations, or they may be mosaic, with TSC2 mutations in the lung and the kidney but not in other organs.

Cloning of a breakpoint cluster region on chromosome 14 in uterine leiomyoma.

A recurrent reciprocal chromosomal translocation, t(12;14)(q15;q24) is frequently observed in uterine leiomyoma. Chromosome 12 breakpoints have been shown to occur in a region of approximately 150 kb that contains the gene for a high mobility group protein (HMGI-C). The breakpoint region on chromosome 14 has not been precisely defined. We have generated a contig of overlapping yeast artificial chromosome (YAC) clones approximately 3 Mb in size. Fluorescence in situ hybridization (FISH) analysis showed that this contig spanned the t(12;14) breakpoints in three uterine leiomyomas and that the breakpoints in these tumors occurred within a 1 Mb region. A 30 kb cosmid spanning one of the breakpoints was isolated to set the stage for identifying regions on chromosome 14 that may cause this region to be a preferential site for chromosomal translocation.

Bicolor fluorescence in situ hybridization on nuclei from formalin-fixed, paraffin-embedded testicular germ cell tumors: comparison with standard metaphase analysis.

Rearrangements of chromosome 12, especially i(12p), are common in testicular germ cell tumors (TGCT). We have developed 12p and 12q chromosome arm-specific painting probes for fluorescence in situ hybridization (FISH) through the use of chromosome microdissection. We developed a method to hybridize these probes to interphase nuclei released from formalin-fixed, paraffin-embedded tumors (PET). In this study, we compared simultaneous bicolor PET FISH painting with metaphase chromosome analysis of fresh tissue from the same tumor. Bicolor PET FISH produced patterns of 12p and 12q hybridization consistent with expectations based on metaphase chromosome analysis; adjoined 12p and 12q regions appeared to detect "normal" chromosome 12s, whereas relatively large isolated regions of 12p were suggestive of i(12p), and small 12p regions probably represent cryptic rearrangements of 12p. Fourteen tumors with successful cytogenetic analyses and available archival material from the same tumor source were selected for study. In a blinded analysis, PET FISH painting assessment was in very close agreement with karyotypic findings in seven subjects, in close agreement in five, and showed less agreement in two. Differences may be due in part to clonal selection during culture for metaphase studies, or regional selection within the tumor. Although PET FISH painting should not replace standard chromosome analysis, this study shows that it can reliably predict chromosome 12 constitution in TGCT, can serve as a useful adjunct to standard cytogenetics when such analysis is unsuccessful, and can provide limited karyotyping of archival materials.

DNA sequence, chromosomal localization, and tissue expression of the mouse proteasome subunit lmp10 (Psmb10) gene.

Proteasomes are nonlysosomal multicatalytic proteases involved in antigen processing. Three of the 10 mammalian proteasome beta subunits (LMP2, LMP7, and LMP10) are induced by IFN-gamma. Two of these (LMP2 and LMP7) are encoded in the major histocompatibility complex of both human (chromosome 6) and mouse (chromosome 17). However, the human homologue of Lmp10, MECL1, is found on chromosome 16. Here we show that in mice, Lmp10 is a single-copy gene localized to chromosome 8, in a region of conserved synteny with human chromosome 16. Sequencing of a 129/SvJ strain genomic clone revealed that the gene has eight exons spanning 2.3 kb. Characterization of a full-length mouse cDNA clone indicates that Lmp10 encodes a protein of 273 amino acids with a calculated molecular weight of 29 kDa and an isoelectric point of 6.86. Northern analysis of Lmp2, Lmp7, and Lmp10 showed expression in heart, liver, thymus, lung, and spleen, but not in brain, kidney, skeletal muscle, or testis.

Identification of multiple chromosome 12 abnormalities in human testicular germ cell tumors by two-color fluorescence in situ hybridization (FISH).

The distribution of segments of the short and long arms of chromosome 12 was distinguished by two-color fluorescence in situ hybridization (FISH) in 27 cytogenetically abnormal testicular germ cell tumors (TGCTs). A 12p-specific probe was developed by chromosomal microdissection and sequence-independent polymerase chain reaction (PCR) amplification and was combined with a commercially available whole-chromosome 12 painting probe. The TGCTs included both i(12p)-positive and i(12p)-negative primary tumors and lymph node metastases from patients in clinical stage I or stage II who were not previously treated with chemotherapy. Rearrangements of the short arm of chromosome 12 and overrepresentation of 12p DNA sequences were found in all cases. In addition, cryptic rearrangements of 12p were found in 39% (7/18) of the i(12p)-positive tumors and in 78% (7/9) of the i(12p)-negative tumors. Only 7% (2/27) of all tumors had cryptic rearrangements of 12q.

High selectivity of polyclonal antibodies against DNA modified by diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxides.

Polyclonal antibodies were developed in New Zealand White rabbits against DNA modified with diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PhDE)-1 (4-hydroxyl and epoxide cis) and B[c]PhDE-2 (4-hydroxyl and epoxide trans). Antiserum developed against B[c]PhDE-2-DNA was stereoselective. In competitive ELISA assays using wells coated with 160 fmol B[c]PhDE-2-DNA adducts, B[c]PhDE-2-DNA gave 50% inhibition at 200 fmol adducts/well. B[c]PhDE-1-DNA required a 10-fold higher amount of adducts/well to give 50% inhibition. Benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA caused only a 30% inhibition even at the highest doses tested (greater than 4000 fmol adducts/well). For antiserum developed against B[c]PhDE-1-DNA, 50% inhibition required 570 fmol B[c]PhDE-1-DNA adducts in wells coated with 100 fmol B[c]PhDE-1-DNA adducts. 7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA and B[c]PhDE-2-DNA were also effective competitors: they caused 50% inhibition at 1900 and 1800 fmol adducts/well respectively. In contrast, benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA gave no inhibition at the highest dose of competitor tested (4050 fmol adducts/well). Antisera from three rabbits immunized with B[c]PhDE-2-DNA demonstrated similar antigen specificities. The properties of these antisera differ from those reported previously for antibodies developed against benzo[a]pyrene-DNA in that they show selectivity for DNA modified by specific hydrocarbon diolepoxides, in one case for B[c]PhDE-2-DNA and in the other for B[c]PhDE-DNA or 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA. The specificity of these antisera will facilitate analysis of the modification of DNA by different polycyclic aromatic hydrocarbon diolepoxides.

Metabolism and cytotoxicity of acetaminophen in hepatocyte cultures from rat, rabbit, dog, and monkey.

Acetaminophen (APAP)-induced cytotoxicity and metabolism were studied in hepatocyte cultures isolated from the rat, rabbit, dog, and monkey. Cytotoxicity was evaluated by morphological examination and by alanine aminotransferase and aspartate aminotransferase released into the cell culture medium. The toxicity results obtained by these two methods were in agreement and can be explained by the biotransformation of APAP in each species. Rat and dog hepatocyte cultures contained the most APAP-sulfate conjugates, while the rabbit, dog, and monkey hepatocyte cultures contained the most APAP-glucuronide conjugates. The percentage of APAP-glutathione conjugate was very low in all species, indicating that either very little of the toxic APAP metabolite, N-acetylbenzoquinoneimine, was formed, or in the species susceptible to N-acetylbenzoquinoneimine-induced cytotoxicity, the glutathione S-transferase activity or the amount of glutathione was low. Rabbit hepatocytes transformed the most APAP during both short and long periods of exposure. Of the four species, the dog hepatocytes exhibited the highest level of APAP-induced cytotoxicity. The sensitivity of dog hepatocytes to APAP may be due to their low conjugating enzyme activity. Rat hepatocytes utilized all three pathways of APAP-biotransformation to prevent APAP-induced cytotoxicity. Monkey hepatocyte cultures had a very large capacity to transform APAP to a glucuronide conjugate and a very high level of glutathione S-transferase activity, and therefore did not exhibit any cytotoxicity. These studies indicate that the competing pathways of APAP conjugation in hepatocyte cultures from different species explain the differences observed in APAP-induced cytotoxicity.

Metabolism and DNA adduct formation of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in fish cell lines in culture.

The metabolic activation of the carcinogens benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) was examined in cell lines derived from bluegill fry (BF-2), rainbow trout (RTG-2) and brown bullhead (BB). All three cell lines metabolized BP (0.5 microgram/ml medium) almost completely to water-soluble metabolites within 120 h, but the maximum amount of BP bound to DNA ranged from only 5 pmol/mg DNA in the BF-2 cells to 17 in the BB cells and 44 in the RTG-2 cells. The major BP-DNA adduct in the BB and BF-2 cells was that formed by reaction of (+)-anti-BP-7,8-diol-9,10 epoxide [(+)anti-BPDE] with deoxyguanosine. This adduct was also present in the RTG-2 cell DNA, but there were larger amounts of unidentified polar BP-DNA adducts. Exposure of the cells to [3H]BP-7,8-diol, a metabolic precursor of (+)anti-BPDE, resulted in binding of 1.5, 12 and 35 pmol BP per mg DNA in the BF-2, BB and RTG-2 cells, respectively. More than 90% of the BP-7,8-diol added to the BF-2 cultures was recovered as a glucuronic acid conjugate, but the RTG-2 cells formed more glutathione conjugates than glucuronide conjugates. The BB cells formed both types of conjugates at a slower rate for more than 75% of the 7,8-diol was recovered unchanged after 24 h. The three cell lines differed in the proportion of a 0.1 microgram/ml dose of DMBA metabolized in 48 h: the values ranged from 47% in the BF-2 cells to 78% in the BB cells and 97% in the RTG-2 cells. The amount of DMBA bound to DNA ranged from 4.7 to 8.6 pmol/mg DNA in the three cell lines: DMBA-3,4-diol-1,2-epoxide (DMBADE) adducts were present in the BB cell DNA, but no significant amounts of DMBADE-DNA adducts were detected in the RTG-2 or BF-2 cell DNA. These results demonstrate that fish cell cultures can activate BP to an ultimate carcinogenic metabolite, (+)anti-BPDE, but the level of binding of this metabolite to DNA is much lower than that which occurs in rodent embryo cell cultures. In BF-2 cell cultures formation of BP-7,8-diol-glucuronide effectively prevents the activation of this diol to (+)anti-BPDE. A substantial proportion of the BP-7,8-diol is also metabolized to glucuronide and glutathione conjugates in BB and RTG-2 cells. DMBA also binds to DNA at very low levels in these fish cell cultures. Thus effective conjugation of diols and their metabolites by fish cell lines appears to greatly reduce metabolic activation of hydrocarbons through the bay-region diol epoxide pathway that predominates in mammalian cell cultures.

Benzo(e)pyrene-induced alterations in the binding of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene to DNA in Sencar mouse epidermis.

Benzo(e)pyrene [B(e)P] cotreatment slightly increases the tumor-initiating activity of benzo(a)pyrene [B(a)P] and greatly decreases the tumor-initiating activity of 7,12-dimethylbenz(a)anthracene (DMBA) in Sencar mice (DiGiovanni et al., Carcinogenesis 3: 371-375, 1982). The effects of B(e)P on the binding of B(a)P and DMBA to Sencar mouse epidermis were investigated using a protocol similar to the mouse skin tumorigenicity studies. After 12 h of exposure to 50 nmol [3H]B(a)P and low or high doses of B(e)P, the level of [3H]B(a)P bound to mouse epidermal DNA increased by 30%. However, after 24 h exposure to 50 nmol [3H]B(a)P and after 12 or 24 h of exposure to 200 nmol [3H]B(a)P, B(e)P had no effect on the amount of [3H]B(a)P bound to DNA. The ration of anti-(the isomer with the epoxide and benzylic hydroxyl on opposite faces of the molecule) B(a)P-7,8-diol-9,10-epoxide [B(a)PDE]-deoxyribonucleoside adducts to syn- (the isomer with the epoxide and benzylic hydroxyl on the same face of the molecule) B(a)PDE-deoxyribonucleoside adducts did not change at either initiating dose of B(a)P or at any time regardless of the dose of B(e)P. After 12 h of exposure to high doses of B(e)P and a 50-nmol initiating dose of B(a)P the level of [3H]B(a)P bound to DNA increased but there was no change in the proportion of particular B(a)PDE-deoxyribonucleoside adducts present. In contrast, B(e)P inhibited the binding of initiating doses of DMBA (5 and 20 nmol) to DNA after 12 and 48 h of exposure to all dose ratios of B(e)P:DMBA tested. The three major adducts, tentatively identified as anti-DMBA-3,4-diol-1,2-epoxide (DMBADE):deoxyguanosine, syn-DMBADE:deoxyadenosine and anti-DMBADE:deoxyadenosine, decreased to the same relative extent as the dose of B(e)P increased. Thus, the effects of B(e)P on the total binding of these hydrocarbons to DNA in epidermis correlate with the cocarcinogenic and anticarcinogenic effects of B(e)P on B(a)P and DMBA, respectively, in a mouse skin initiation-promotion assay. These results indicate that the mechanism of the co- or anticarcinogenic action of hydrocarbons such as B(e)P involves alteration of the binding of carcinogenic hydrocarbons to DNA. They also suggest that measurement of carcinogenic hydrocarbon-DNA adducts formed during cotreatment with other hydrocarbons will provide a rapid method for predicting the co- or anticarcinogenic effect of the other hydrocarbons.