[Investigation on the 3 D geometric accuracy and on the image quality (MTF, SNR and NPS) of volume tomography units (CT, CBCT and DVT)]. / Untersuchung zur geometrischen 3-D-Genauigkeit und zur Bildqualität (MTF, SRV und W) von Volumentomografie-Einrichtungen (CT, CBCT und DVT).

PURPOSE: The study aims at investigating how far image quality (MTF and NPS) differs in between CT, CBCT and DVT units and how far the geometrical 3 D accuracy and the HU calibration differ in respect to surgical or radio therapeutic planning. MATERIALS AND METHODS: X ray image stacks have been made using a new designed test device which contains structures for measuring MTF, NPS, the 3 D accuracy and the Hounsfield calibration (jaw or skull program). The image stacks of the transversal images were analyzed with a dedicated computer program. RESULTS: The MTF values are correlated with the physical resolution (CT and DVT) and are influenced by the used Kernel (CT). The NPS values are limited to an intra system comparison due to the insufficient HU accuracy. The 3 D accuracy is comparable in between the system types. CONCLUSIONS: The values of image quality are not yet correlated with dose values: NPS. Investigations to an appropriate dosimetry are ongoing to establish the ratio between dose and image quality (ALARA principle). No fundamental difference between the systems can be stated in respect radio therapeutic planning: improper HU calibration accuracy in CBCT and DVT units. The geometric 3 D accuracy of high performance DVT systems is greater than that of CT Systems.

SU-E-I-05: Dose Reduction in Head CBCT Examinations After Protocol Optimization.

PURPOSE: CBCT systems for head examinations especially in otorhinolaryngology offer better than required image quality at relatively low purchasing costs. Due to this fact the number of dedicated head scanners increases steadily. Regarding image quality in relation to patient dose these systems are frequently not optimized. Examination parameter optimization is an iterative process, which should be done carefully by physicians in cooperation with physicists. After a successful optimization, Monte Carlo Simulations can be used to quantify the dose reduction to the patient. METHODS: Images of an anatomic head phantom were obtained using different settings of kVp, mAs and rotation angle (360°, 210°). The resulting images were anonymized and the examination parameters were removed. Radiologists and otorhinolaryngologists evaluated these images and rated them into excellent image quality, good image quality, bad image quality and not readable. Based on the ratings new parameters for the adequate image quality were set. The dose reduction was calculated using Monte Carlo simulations. RESULTS: The organ dose of radiation sensitive organs was reduced significantly. The dose reductions compared to the standard settings are: eyes: 85%, eye lenses: 88%, salivary glands: 40%, thyroid: 60%, blood vessels: 40%, brain: 60%, teeth: 80% and tonsils: 65%. Dose reduction was best, when a short scan (210° rotation angle) was used, where the eyes are not in the direct exposure field. CONCLUSIONS: The results show the potential for dose optimizations in otorhinolaryngological CBCT examinations compared to standard vendor settings without loss in diagnostic information. Furthermore this study points out the great opportunities that Monte Carlo-based dose-calculations methods offer to quantify the increase of dose efficiency after examination protocol optimizations. It could also be shown that CBCT has advantages when doing short scan mode around the rear head. This protects radiation sensitive organs such as the eye lenses from being directly radiated.

Bufuralol metabolism by guinea pig adrenal and hepatic microsomes.

Previous investigations demonstrated that CYP2D16 was expressed at high levels in guinea pig adrenal microsomes. The studies presented here were done to determine whether adrenal metabolism of bufuralol (BUF), a model CYP2D substrate, was similar to that in the liver. Guinea pig adrenal microsomes converted BUF to 1'-hydroxybufuralol (1'-OH-BUF) as the major metabolite and smaller amounts of a compound identified as 6-hydroxybufuralol (6-OH-BUF). In contrast, 6-OH-BUF was the major product formed by hepatic microsomal preparations. The apparent Km values were similar for 1'-OH-BUF and 6-OH-BUF production in each tissue. Quinidine, a selective CYP2D inhibitor, decreased the production of both BUF metabolites equally in liver and adrenal microsomes. Cortisol also caused equivalent decreases in the rates of 1'-OH-BUF and 6-OH-BUF formation by adrenal microsomes, but had no effect on hepatic BUF metabolism. Although both BUF metabolites may be produced by CYP2D16, unknown factors appear to effect some differences in the catalytic characteristics of BUF metabolism in adrenal and liver. The large amount of 6-OH-BUF produced distinguishes BUF metabolism in guinea pigs from that in other species previously studied.

Suppression of lipid peroxidation in adrenal microsomes following ACTH administration to guinea pigs.

Previous studies demonstrated high levels of lipid peroxidation (LP) in the guinea pig adrenal cortex. The present studies were done to determine if adrenal LP activity was influenced by ACTH, the major hormonal regulator of the gland. Guinea pigs were treated with ACTH for 1, 3 or 7 days. In addition, some guinea pigs received ACTH for 7 days and were killed 3 or 7 days later. After treatment, adrenal microsomal fractions were prepared and incubated in vitro with 1 mM ferrous sulfate to initiate LP. ACTH treatment caused a progressive decrease in adrenal LP; activity was almost totally inhibited within 3 days. The inhibitory effects of ACTH on LP were dose-dependent. Following cessation of ACTH treatment, adrenal LP gradually returned toward control levels. Microsomal concentrations of linoleic acid, a major substrate for adrenal LP, were increased by ACTH administration and then also returned to control levels after cessation of treatment. There were no significant changes in adrenal alpha-tocopherol or beta-carotene concentrations resulting from ACTH treatment. The results indicate that ACTH has a role in the regulation of adrenal LP. The actions of ACTH cannot be attributed to an increase in adrenal content of the antioxidants, alpha-tocopherol and beta-carotene, or to a decrease in LP substrate. The actions of ACTH to inhibit LP may contribute to an increase in adrenal hormone production by protecting steroidogenic enzymes from peroxidative degradation.

Maturational changes in CYP2D16 expression and xenobiotic metabolism in adrenal glands from male and female guinea pigs.

CYP2D16 is expressed at high levels in the zona reticularis (ZR) of guinea pig adrenal glands and contributes to adrenal metabolism of xenobiotics. Studies were done to evaluate the effects of age and gender on adrenal CYP2D16 expression and xenobiotic metabolism. In both male and female guinea pigs at 1, 7, 14, or 30 weeks of age, in situ hybridization and immunohistochemistry confirmed that CYP2D16 was highly localized to the ZR of the adrenal gland. The steroidogenic P450 isozyme, CYP17, by contrast, was expressed in both the zona fasciculata and ZR. The intensity of CYP2D16 staining was not age- or gender-dependent. However, the proportion of each adrenal gland comprised by ZR and thus expressing CYP2D16 increased with aging in both sexes and was greater in males than in females. The rates of metabolism of bufuralol, a CYP2D-selective substrate, by adrenal microsomal preparations generally correlated with the amount of ZR (and CYP2D16) in the gland. Thus, adrenal xenobiotic-metabolizing activities were greater in males than in females at all ages and increased with aging in males. However, the rates of bufuralol metabolism declined in sexually mature females (14 weeks) from the levels found in prepubertal females (7 weeks) and then increased markedly in retired breeders (30 weeks), suggesting an inhibitory effect of estrogens on enzyme activity. The results indicate that the age and gender differences in adrenal CYP2D16 content are largely determined by differences in the size of the ZR rather than the concentrations of CYP2D16 within cells of the ZR. However, adrenal xenobiotic-metabolizing activities in females seem to be further modulated by an inhibitory effect of estrogens.

Bimodal expression of CYP2D16 in the guinea pig adrenal cortex.

Prior studies revealed an apparent bimodal distribution of adrenal xenobiotic-metabolizing activities in outbred guinea pigs. High activities were characteristic of most animals but a minority had very low rates of metabolism. The present studies were done to determine the basis for this distribution. Among the enzymatic activities abundant in guinea pig adrenal microsomes is bufuralol 1'-hydroxylation, a marker for CYP2D isozymes. Western blot analyses revealed that adrenal expression of CYP2D16, the guinea pig CYP2D analogue, was highly correlated with xenobiotic-metabolizing activities. Of 12 animals studied, 4 had low levels of adrenal CYP2D16 expression and correspondingly low bufuralol 1'-hydroxylase and benzphetamine N-demethylase activities. In contrast, adrenal expression of steroidogenic P450 isozymes (CYP17 and CYP21) and the corresponding steroid hydroxylase activities were similar in all animals. There was also no evidence of polymorphism in hepatic xenobiotic metabolism. The results suggest that the bimodal expression of adrenal CYP2D16 is at least partly responsible for the variability in adrenal xenobiotic metabolism, and that this pattern of expression is not applicable to other adrenal P450 isozymes or to hepatic CYP2D16. Further studies are needed to determine if genetic polymorphism is responsible for the mode of adrenal CYP2D16 expression.

Strain differences in adrenal microsomal steroid metabolism in guinea pigs.

We recently reported that CYP2D16, a xenobiotic-metabolizing P450 isozyme, was expressed at higher levels in adrenal microsomes from inbred Strain 13 guinea pigs than in those from outbred English Short Hair (ESH) animals. Studies were done to determine if there also were strain differences in adrenal microsomal steroid metabolism. In both inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zone preparations of the adrenal cortex, 21-hydroxylase activities were greater in microsomes from ESH than from Strain 13 guinea pigs. By contrast, 17alpha-hydroxylase activities were similar in the two strains. In both strains, 21-hydroxylase activities were greater in inner than outer zone microsomes, but the opposite was found for 17alpha-hydroxylase activities (outer>inner). Northern and Western analyses revealed higher levels of CYP21 mRNA and protein in adrenals from ESH than Strain 13 guinea pigs, but there were no strain differences in CYP17 mRNA or protein concentrations. Despite the zonal differences in adrenal 17alpha-hydroxylase and 21-hydroxylase activities, CYP17 and CYP21 mRNA and protein levels were similar in the inner and outer zones within each strain of guinea pig. The results demonstrate strain differences in microsomal steroid metabolism that are explained by differences in CYP21 expression. By contrast, the zonal differences in steroid hydroxylase activities may be attributable to post-translational mechanisms.

Expression of a vitamin D-regulated gene (VDUP-1) in untreated- and MNU-treated rat mammary tissue.

Previous studies showed that the expression of an mRNA corresponding to VDUP-1 was decreased within MNU-induced rat mammary tumors. RNA from mammary tissue was DNase treated and reverse transcribed and the resulting cDNA was amplified using primers designed to amplify VDUP-1 (382 bp fragment) and glyceraldehyde-6-phosphate dehydrogenase (GAPDH) (416 bp fragment). Analysis of mammary cDNA derived from untreated or MNU-treated rats indicated that VDUP-1 expression within tumor tissue was significantly decreased, a finding which agrees with previous Northern blotting experiments. The differential expression was confirmed in tissue sections using an antisense VDUP-1 riboprobe for in situ hybridization studies which demonstrated that VDUP-1 staining in all cell types within tumor tissue was greatly decreased. VDUP-1 mRNA was expressed to a greater extent within epithelial cells and to a much lesser extent within stromal cells, including endothelial cells, in untreated mammary tissue. A significant decrease in VDUP-1 expression was detected as early as six weeks after MNU treatment, before tumors had formed. Bilateral ovariectomy did not alter VDUP-1 expression in untreated mammary tissue and ovariectomy prior to MNU treatment prevented tumor formation, as well as the associated decrease in VDUP-1 expression. The relative expression of VDUP-1 was higher in lung tissue than in adrenal, heart, kidney, liver, mammary, muscle, and ovary. Treatment of a cell line derived from an MNU-induced rat mammary tumor (MNU cells) with 1,25-dihydroxyvitamin D3 resulted in a significant increase in VDUP-1 expression and also inhibited cell growth in the absence of serum. The data are consistent with a role for VDUP-1 in mediating the inhibitory effects of 1,25-dihydroxyvitamin D3 on tumor cell growth.

Effects of ACTH administration on zonation of the guinea pig adrenal cortex.

Experiments were done to determine the actions of ACTH on the morphologic and functional characteristics of the zona fasciculata (ZF) and zona reticularis (ZR) in the guinea pig adrenal cortex. In control guinea pigs, a number of morphologic differences distinguished the ZF from the ZR, including the presence of far more lipid in the ZF than in the ZR. Treatment with ACTH decreased the lipid droplet content of the ZF cells, equalizing the amount of lipid in the two zones. Other morphologic differences between the ZF and ZR were also diminished by ACTH treatment. Immunohistochemical analyses indicated that CYP17 protein was found in both the ZF and ZR in control animals, but with greater immunostaining intensity in the ZF. The enzyme protein distribution corresponded with higher 17alpha-hydroxylase activity in the ZF than in the ZR. After ACTH treatment, the intensity of staining and enzyme activities in the two zones were similar, attributable largely to increases in the ZR. In situ hybridization-and immunohistochemistry showed that in control animals CYPD216 was highly expressed in the ZR but not in the ZF. ACTH treatment dramatically reduced the intensity of CYP2D16 mRNA and protein staining in the ZR. Bufuralol 1'-hydroxylase activity, a marker for CYP2D subfamily members, was also decreased significantly in the ZR by ACTH treatment. The data indicate that administration of ACTH to guinea pigs has opposite effects on the expression of CYP17 and CYP2D16 in the ZR, and diminishes or eliminates some of the structural and functional differences between the ZF and ZR. The results suggest a role for ACTH in establishing and maintaining adrenocortical zonation.

Mitogenic responses of rat nasal epithelium to hexamethylphosphoramide inhalation exposure.

Hexamethylphosphoramide (HMPA) is a rat nasal carcinogen that induces squamous cell carcinomas in the anterior portions of the nasal cavity following chronic inhalation exposures as low as 50 ppb. These tumors may arise as a result of P-450-mediated release of formaldehyde (HCHO), a known rat nasal carcinogen. The goal of this research was to investigate early responses of the nasal epithelium to inhaled HMPA. Rats were exposed nose-only to approximately 3 ppm HMPA for 6 h, and killed 18, 48, 96 or 144 h post-exposure. In a separate study, rats were exposed nose-only for 6 h for 1, 2, 3, or 5 consecutive days and killed 18 or 96 h post-exposure. With both single and repeated doses of HMPA, there was no evidence of cytotoxicity in the anterior nose. Olfactory degeneration and necrosis of the dorsal meatus, Bowman's glands and tips of the ethmoid turbinates increased in severity with repeated exposures to HMPA. Cell proliferation was assessed in levels of nasal tissue that included regions of squamous, respiratory, transitional and olfactory epithelium. Regional induction of cell proliferation was measured by BrdU incorporation, and reported as the number of labeled cells/mm basement membrane. At 18 h after a single exposure, there was an increase in cell proliferation in squamous epithelium, which returned to control levels within 48 h. A transitory increase in cell proliferation was observed regions of respiratory and transitional epithelium, although the response of each tissue, in terms of magnitude and peak time of response post-exposure, also differed. Along the dorsal meatus in Level 9, olfactory labeling initially decreased, returned to control levels by 96 h, but again declined at 144 h post-exposure. In repeat dose studies, the squamous epithelium response was variable 18 h post-exposure. For respiratory and transitional epithelium, increased cell proliferation 18 h post-exposure was correlated with increased dose (exposure) of HMPA. Cell proliferation responses following two or more exposures returned to near control levels within 96 h post-exposure. In conclusion, HMPA induced cell proliferation, but not cytotoxicity, in the anterior nose at approximately 3 ppm. These data suggest that HMPA induces proliferative, perhaps mitogenic, responses in the nasal epithelium, and this response may facilitate the fixation of low level genetic damage induced by liberated HCHO.

Localization of CYP2D16 in the guinea pig adrenal cortex by immunohistochemistry and in situ hybridization.

Recent reports indicate that the cytochrome P450 isozyme, CYP2D16, is expressed at high levels in the inner regions of the guinea pig adrenal cortex and may contribute to xenobiotic and/or steroid metabolism in the gland. In the present studies, immunohistochemical and in situ hybridization techniques were employed to definitively establish the localization of CYP2D16 within the adrenal cortex. In male guinea pigs of various ages, CYP2D16 protein and mRNA were highly localized to the zona reticularis (ZR); none was detectable in the zona fasciculata (ZF), zona glomerulosa (ZG) or the medulla. In contrast, the steroidogenic P450 isozyme, CYP17, was distributed throughout the ZF and ZR. From the earliest stages of development of the ZR, CYP2D16 staining was intense. As guinea pigs aged, the ZR progressively enlarged and comprised a proportionately greater amount of the cortex. At all ages, CYP2D16 was uniformly distributed throughout only the ZR. Coinciding with the age-related growth of the ZR and increase in adrenal CYP2D16 content was an increase in adrenal xenobiotic-metabolizing activity. The results establish that CYP2D16 has an intraadrenal localization that is unique among P450 isozymes, suggesting novel regulatory mechanisms and indicating that CYP2D16 may serve as a specific marker for ZR cells. The increase in CYP2D16 expression with age probably accounts for increasing levels of xenobiotic metabolism and may also contribute to an increase in intraadrenal cortisol degradation in older animals.

Strain differences in adrenal CYP2D16 expression in guinea pigs. Relationship to xenobiotic metabolism.

Experiments were done to determine the mechanisms responsible for differences in adrenal microsomal xenobiotic metabolism between Strain 13 and English Short-Hair (ESH) guinea pigs. The rates of adrenal xenobiotic metabolism (bufuralol 1'-hydroxylase, benzo[a]pyrene hydroxylase, benzphetamine N-demethylase) were 2-3 times greater in microsomes from the Strain 13 animals. In both strains, xenobiotic-metabolizing activities were far greater in the inner zone (zona reticularis) than in the outer zones (zona fasciculata and zona glomerulosa) of the adrenal cortex. Northern blot analyses of total adrenal RNA with a CYP2D16 cDNA as the probe revealed significantly greater amounts of CYP2D16 mRNA in the Strain 13 guinea pigs. In addition, SDS-PAGE and Western blotting of adrenal microsomes demonstrated higher concentrations of CYP2D16 protein in Strain 13 than in ESH animals. Expression of CYP2D16 was predominantly in the inner zone of the adrenal, coinciding with the major site of xenobiotic metabolism. The results demonstrated higher levels of expression of CYP2D16 in adrenal glands from Strain 13 than from ESH guinea pigs, which may account for the strain differences in adrenal xenobiotic metabolism. Strain 13 guinea pigs should serve as a good experimental model for further studies on the regulation of adrenal CYP2D16.

Differential effects of adrenocorticotropin in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex.

Studies were performed to compare the effects of ACTH treatment in vivo on cytochromes P4502D16 and P450c17 in the guinea pig adrenal cortex. In untreated animals, CYP2D16 protein and messenger RNA (mRNA) expression as well as xenobiotic-metabolizing activities (bufuralol 1'-hydroxylase, benzphetamine N-demethylase, and benzo(a)pyrene hydroxylase) were far greater in the inner (zona reticularis) than the outer (zona fasciculata plus zona glomerulosa) zones of the cortex. ACTH treatment for 3 or 7 days significantly decreased the rates of xenobiotic metabolism in both the inner and outer adrenal zones. Western and Northern blot analyses revealed that adrenal CYP2D16 protein and mRNA concentrations were significantly decreased by ACTH. In contrast to its inhibitory effects on CYP2D16, ACTH treatment increased steroid 17 alpha-hydroxylase activity in the adrenal inner zone, but did not affect outer zone activity. Microsomal CYP17 protein concentrations were not affected by ACTH despite increases in CYP17 mRNA levels in both zones. The results indicate that ACTH causes down-regulation of adrenal CYP2D16, probably at the transcriptional level. Thus, modulation of CYP2D16 by ACTH is opposite that for the steroidogenic P450 isozymes, suggesting unique regulatory mechanisms. In addition, the data suggest that posttranscriptional mechanisms contribute to ACTH regulation of 17 alpha-hydroxylase activity in the guinea pig adrenal cortex.

Alteration of gene expression in rat mammary tumors induced by N-methyl-N-nitrosourea.

Rodents are susceptible to the effects of chemical carcinogens and have been widely used in the study of mammary-gland carcinogenesis. However, little information is available regarding specific phenotypic changes that occur during mammary-gland carcinogenesis. In this study, subtraction hybridization was used to identify specific genes whose expression in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors had been altered. mRNA isolated from normal rat mammary tissue and tumors induced by treatment of 50-d-old female rats with MNU (50 mg/kg) was used to produce normal and tumor cDNA libraries. Total inserts prepared from each cDNA library were used to produce a subtracted tumor-normal probe. Differential screening of the tumor library with the subtracted probe and normal cDNA yielded 20 clones that appeared to be differentially expressed. Northern analysis of mRNA isolated from normal mammary tissue and tumor tissue confirmed that four of these clones were differentially expressed. The expression of clones 4 and 15 was greatly increased (13-fold and tenfold, respectively) in most MNU-induced mammary tumors, whereas the expression of clones 10 and 27 was decreased (13-fold and fourfold, respectively). Sequence analysis revealed that clones 15 and 27 were highly homologous to calcyclin and a cDNA isolated from HL-60 cells, respectively. The differential expression of clones 4 and 10 was due to the presence within these clones of retroviral sequences and a fragment of transferrin, respectively. These clones may represent markers useful for studying the development of MNU-induced mammary-gland neoplasias.

Expression and zonal distribution of CYP2D16 in the guinea pig adrenal cortex: relationship to xenobiotic metabolism.

We recently cloned a CYP2D subfamily member (CYP2D16) from a guinea pig adrenal cDNA library and investigated the expression of CYP2D16 in the guinea pig adrenal cortex and its relationship to adrenal xenobiotic metabolism. A modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed three major bands in the molecular mass range of cytochromes P450 in guinea pig adrenal microsomes. Two of the bands were immunoreactive with anti-CYP17 (54 kDa) or anti-CYP21 (52 kDa) antibody. The third band (50 kDa) was immunoreactive with antibody raised against CYP2D1 and with anti-CYP1A1/1A2 antibody. Microsequencing of the 50-kDa band yielded an amino-terminal sequence of 38 amino acids identical to that deducted from the CYP2D16 cDNA. In addition, Northern blot analyses indicated the CYP1A1 was not expressed in the adrenal gland, suggesting that only CYP2D16 composed the microsomal 50-kDa band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses demonstrated greater expression of CYP2D16 in microsomes from the inner zone (zona reticularis) of the adrenal cortex than from the outer zones, coinciding with the major site of adrenal xenobiotic metabolism. Bufuralol-1'-hydroxylase activity, a marker for CYP2D isozymes, was also greater in inner- than in outer-zone microsomal preparations and was highly correlated with CYP2D16concentrations. Northern blot analysis with a full-length CYP2D16 cDNA as the probe gave strong bands with adrenal inner zone RNA preparations and relatively weak bands with outer zone RNA. CYP2D16 mRNA was also detectable in liver and kidney RNA preparations, but at lower levels than in the adrenal inner zone, and it was not detectable in testes, lung, intestines, or heart. Overall, the results demonstrate that CYP2D16 is expressed at highest levels in the inner zone of the guinea pig adrenal cortex and suggest a major role for this isozyme in adrenal xenobiotic metabolism.

Evidence for a single non-arachidonic acid-specific fatty acyl-CoA synthetase in heart which is regulated by Mg2+.

Previous reports indicated that arachidonic acid is incorporated into the isolated perfused rabbit heart in preference to other fatty acids, and that incorporation of arachidonic acid, but not other fatty acids, is inhibited during Mg2+ depletion. In this study, we have not been able to demonstrate an arachidonic acid-specific fatty acyl-CoA synthetase in rat or rabbit heart by hydroxyapatite chromatography. Kinetic evidence was consistent with a single enzyme, as the slopes of pseudo-Hill plots were not significantly different from -1. The single fatty acyl-CoA synthetase present appears to prefer C18:0 unsaturated fatty acids to arachidonate, and had about the same affinity for C10:0 -C14:0 saturated fatty acids as for arachidonate. At 35 microM arachidonate, enzyme velocity increased as the total Mg2+ was increased from 3 to 80 mM. Calculated [MgATP] indicated that the MgATP complex was not rate-limiting. At low concentrations, Mn2+ and Ni2+ supported activity, but Cu2+ and Zn2+ did not. Low Ca2+ concentrations activated only oleic acid conversion. Kinetic analysis indicated that the Vmax of the enzyme was increased with increasing concentrations of ionized Mg2+ for both oleic acid and arachidonic acid. The data are constant with the hypothesis that Mg2+ has a direct effect on fatty acyl-CoA synthetase activity, and suggest that preference for oleic acid and arachidonic acid can be influenced by the ionic milieu.

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs.

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration of guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17 alpha-hydroxylase and 21-hydroxylase activities were decreased to 20-25% of control values by the higher dose of ABT. Mitochondrial 11 beta-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3 beta-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.

O6-methylguanine-induced replication blocks.

The ability of Klenow polymerase I, phage T7 polymerase (Sequenase), human polymerase alpha, and human polymerase beta to synthesize past (bypass) O6-methylguanine (O6-meG) lesions was studied in the presence of MgCl2 and MnCl2. An end-labeled 16-mer primer was annealed to the 3' end of gel-purified oligodeoxyribonucleotide templates (45-mers), each containing a single O6-meG in place of one G in the sequence -G1G2CG3G4T-. Extension products were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. A fraction of the products extended by Klenow fragment terminated either opposite or one base before O6-meG located at sites 1 and 3. Termination occurred primarily one base before O6-meG located at sites 2 and 4. The remaining fractions that bypassed the lesions represented full-length product. In control reactions, the O6-meG-containing templates were annealed with complementary 45-mers, repaired with O6-alkylguanine DNA-alkyltransferase, annealed with an excess of labeled primer, and extended by Klenow fragment. Full-length extension of > 90% was observed with each template. Primer extension past O6-meG by DNA polymerase alpha and Sequenase was partially blocked in a manner which varied with the site of O6-meG in the template while primer extension by DNA polymerase beta was completely blocked (< 2% full length extension) with O6-meG at sites 1-4. Substitution of MnCl2 for MgCl2 in the reaction mixture greatly increased the bypass of O6-meG by Klenow fragment and DNA polymerase alpha but not Sequenase or DNA polymerase beta. The increased ability of Klenow fragment to bypass O6-meG in the presence of MnCl2 was found to result from an increased incorporation of G (O6-meG at sites 1 and 2) and A (O6-meG at sites 1, 2, and 3) opposite the lesion. The results indicate that O6-meG can block in vitro polymerization by several DNA polymerases and are consistent with the observed cytotoxic effects of methylating agents on mammalian cells.

Molecular cloning and sequencing of a guinea pig cytochrome P4502D (CYP2D16): high level expression in adrenal microsomes.

Studies were done to characterize a guinea pig adrenal microsomal P450 that had been linked with xenobiotic metabolism in the inner zone of the gland. N-terminal amino acid sequencing of the isolated protein revealed homology with members of the CYP2D subfamily. A human CYPD2D6 cDNA probe was used to screen a guinea pig adrenal cDNA library and a full-length clone was obtained having an open reading frame encoding a 500 amino acid protein. The sequence was found to be highly homologous with all members of the CYP2D subfamily and was designated CYP2D16. The N-terminal sequence of 38 amino acids obtained from the protein microsequencing was identical to that deduced from the nucleotide sequence of the cloned CYP2D16. Northern blot analysis confirmed that CYP2D16 is expressed at high levels in the inner zone of the guinea pig adrenal cortex. The results suggest that CYP2D16 may account, at least in part, for the high rates of xenobiotic metabolism in the guinea pig adrenal.

Inhibition of adrenal cytochromes P450 by 1-aminobenzotriazole in vitro. Selectivity for xenobiotic metabolism.

Studies were done to determine the effects of a P450 suicide inhibitor, 1-aminobenzotriazole (ABT), on adrenal steroid and xenobiotic metabolism. Incubation of guinea pig adrenal microsomes with ABT plus an NADPH-generating system caused a time-dependent decline in total P450 concentrations. The maximal decrease in P450 levels was approximately 35% and was accompanied by an equimolar decrease in heme content. Western blot analyses indicated that ABT had no effect on P450 apoprotein levels. Benzphetamine (BZ) N-demethylase and benzo[a]pyrene (BP) hydroxylase activities were inhibited almost completely by microsomal incubations with ABT. In contrast, neither steroid 17 alpha-hydroxylase nor 21-hydroxylase activity was affected by ABT. The steroid-induced type I spectral change in adrenal microsomes also was not affected by ABT, whereas that induced by BZ was eliminated. Similar studies with adrenal mitochondria indicated that ABT had no effect on mitochondrial P450 concentrations or on mitochondrial steroid metabolism. The results demonstrate that the in vitro actions of ABT on adrenal cytochromes P450 are highly selective for those isozymes that catalyze xenobiotic metabolism. Therefore, ABT should serve as a useful probe for further characterization of adrenal xenobiotic-metabolizing P450 isozymes.