Species specificity affects the choice of S9 preparations for use in the hamster embryo cell transformation system

Summary Before their use as a source of carcinogen-activating enzymes in the hamster embryo cell transformation assay, liver, kidney, lung, and small intestine S9 fractions from Syrian golden hamsters and Sprague-Dawley rats were evaluated for toxicity to hamster embryo target cells. Sprague-Dawley rat liver and kidney S9 were highly toxic to the hamster embryo cells (90 to 100%). When retested at lower concentrations these tissue fractions were still quite toxic (up to 75%). In contrast, hamster liver and kidney S9 were considerably less toxic (14 to 25%). The S9 preparations were also evaluated for their ability to metabolizeN-2-acetylaminofluorene to 2-aminofluorene andN-hydroxy-acetylamino-fluorene, products that transform hamster embryo cells. Large amounts ofN-hydroxy-acetylaminofluorene were formed in the presence of preparations from hamster liver and small intestine, whereas kidney and lung S9 fractions were considerably less active. No detectable levels ofN-hydroxy-acetylaminofluorene were formed after incubation ofN-2-acetylaminofluorene with any of the rat S9 preparations. High levels of deacetylase activity were found in hamster liver and small intestine S9 fractions, at least eightfold higher than those obtained from equivalent rat preparations. Hamster kidney and lung S9 fractions showed low levels of deacetylase activity. There was no detectable activity in equivalent preparations from rats. When tested withN-2-acetylaminofluorene in the hamster embryo cell clonal transformation system, transformed colonies were obtained with hamster liver S9, with and without an external NADPH-generating system. This work was supported by Contract N01-CO-75380 with the National Cancer Institute, NIH, Bethesda, MD 20205.     

Inhibition of the mutagenic activity of some heterocyclic dietary carcinogens and other mutagenic/carcinogenic compounds by rat organ preparations

The mutagenic activity of some dietary mutagens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), was inhibited in the Salmonella-plate test preincubated with heat-inactivated rat intestinal preparations. A similar inhibition was observed by preincubating intestinal preparations with 2-acetylaminofluorene (AAF) and benzo[a]pyrene (B[a]P). The effect was not specific for small intestine and was also obtained with spleen, liver, lung, colon and stomach preparations. Mutagenic activity was not inhibited by beef muscle proteins. Lipids extracted from intestinal mucosa preparations were equally effective as inhibitors of the mutagenic activity. Lipid fractions from intestinal mucosa were capable of inhibiting the formation of activated IQ by mammalian S9, and other components of the intestinal preparations were able to bind the promutagens and their active metabolites. The mutagenic activity of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (metronidazole) and of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was also inhibited by intestinal preparations, but not by their lipid fractions. A binding of IQ to intestinal preparations was also demonstrated with HPLC techniques. The data indicate that tissue components may reduce the mutagenic activity of chemicals by interfering with the activation process and by reducing the concentration of the promutagens and their active metabolites at target sites.     

Evaluation of a methodology for the use of preparations from rat small intestine in the Salmonella/microsome assay

A methodology is evaluated for the use in the Ames assay of a microsomal metabolising system derived from villous tip cells of rat small intestine. The procedure involved high frequency vibration of everted gut segments followed by gentle lysis and homogenisation. This technique, which has previously been shown to result routinely in high levels of cytochrome P450 and linked enzymes, has now been investigated for its ability to yield preparations capable of activating several promutagens in the Salmonella/plate incorporation test. The data obtained have been compared with results observed with standard rat liver metabolising fractions. In the presence of intestinal microsomes, 2-aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene, aflatoxin B1, benzo[a]pyrene and cyclophosphamide all caused dose-related increases in revertants, the maximum yields of which were lower than those detected with liver microsomes or S9 mix. These and other differences in dose-responses have been discussed in relation to the levels of microsomal protein and cytochrome P450 plated and with respect to the activities of relevant enzymes in the tissue extracts.     

Lysolecithinase activity in subcellular fractions of rat organs.

Lysolecithinase activity was measured in subcellular fractions of rat liver, kidney, lung and intestine and compared to similar findings in brain. To obtain optimal assay conditions, each fraction was subjected to a kinetic analysis in the absence and presence of albumin. Among the particulate preparations, lysolecithinase activity of the intestine exceeded by far similar fractions of other organs. Among the soluble fractions, the 100,000xg supernatant of lung had the highest activity. Under the assay conditions used, most of the lysolecithinase activity of all organs was particulate.     

Characterization of the rates of testosterone metabolism to various products and of glutathione transferase and sulfotransferase activities in rat intestine and comparison to the corresponding hepatic and renal drug-metabolizing enzymes

Metabolism of testosterone to various products (catalyzed by several different CYP isozymes) and the activities of phenol sulfotransferase (pST) and glutathione transferase (GST) in S9 fractions prepared from the mucosa of the duodenum, jejunum, ileum, caecum and upper and lower colon of male Sprague-Dawley rats were determined and compared to the corresponding hepatic and renal activities. Incubation of the S9 fraction prepared from the jejunum with testosterone and NADPH resulted in the formation of 2alpha-, 6alpha-, 6beta- and 16alpha-hydroxytestosterone and androstenedione at rates that were 1.6, 24, 1.3, 0.6 and 1.3%, respectively, of the corresponding hepatic values. The production of 2alpha-hydroxytestosterone was catalyzed only by the preparations from the duodenum and jejunum; whereas 6alpha-, 6beta- and 16alpha-hydroxytestosterone and androstenedione were produced in all regions of the intestine. In the case of the rat kidney, the rates of formation of the different testosterone metabolites were between 0.6 and 35% of the corresponding liver activity. The activity of glutathione transferase was approximately 12-26% of the corresponding hepatic activity throughout the intestine. The highest activity of phenol sulfotransferase was observed in the lower colon (almost 6% of the liver activity) and the lowest activity in the duodenum (1%). The renal activities of GST and pST were 70 and 1%, respectively, of the corresponding liver values. In summary, the metabolism of testosterone and the activities of GST and pST in rat intestine are generally low to very low in comparison to the corresponding activities in rat liver. In most cases, these activities are present throughout the entire intestine and not restricted to a particular portion(s) of this organ.     

Evidence for a second arylhydroxamic acid acyltransferase species in the small intestine of the rat.

The small intestine of the Sprague-Dawley rat has been shown to contain two species of arylhydroxamic acid acyltransferases. These enzymes were separable by gel filtration on Sephadex G-100. The smaller species had the mobility of rat liver acyltransferase and was precipitated with antiserum directed against the liver enzyme. The larger species was not precipitated with this antiserum. These species differ in their relative abilities to utilize N-hydroxy-N-2-acetylaminofluorene (N-hydroxy-AAF) and N-hydroxy-N-4-acetylaminobiphenyl (N-hydroxy-AABP) as substrates, and in their inhibition by non immune serum.     

Metabolic activation of 2-aminofluorene, 2-acetylaminofluorene and N-hydroxy-acetylaminofluorene to bacterial mutagens with mussel (Mytilus galloprovincialis) and carp (Cyprinus carpio) subcellular preparations

1. The mode of activation of 2-aminofluorene (AF), 2-acetylaminofluorene (AAF) and N-hydroxy-acetylaminofluorene (OH-AAF) to Salmonella typhimurium TA 98 mutagens was investigated in subcellular fractions from the digestive gland of the mussel Mytilus galloprovincialis and from the liver of carp Cyprinus carpio. 2. In carp liver microsomes the activation of OH-AAF was due to very active deacetylase, in contrast to undetectable deacetylase-dependent activation in mussel microsomes. 3. AF and AAF are activated in mussel microsomes exclusively by a noninducible FAD-containing monooxygenase, whereas in carp microsomes in addition deacetylase and inducible cytochrome P-450 monooxygenase are involved. 4. N,O-Acetyltransferase, sulfotransferase and paraoxon sensitive cytosolic enzyme (PSCE) are involved in activation of OH-AAF, AF and AAF in both carp and mussel cytosols. 5. The metabolic activation of OH-AAF, AF and AAF to bacterial mutagens found in carp liver is similar to that described in livers of experimental mammalian species and strikingly different from the mode of activation found in mussel digestive gland.     

Mutagenicity of benzo(a)pyrene in uninduced tissues from BALB/c mice and Sprague-Dawley rats as an index of possible health risks using the Salmonella mutagenicity assay

The mutagenicity of benzo(a)pyrene [B(a)P] in uninduced tissues from Sprague-Dawley rats and BALB/c mice resulted in no age, sex or tissue-related differences when S9 preparations from lung, kidney and spleen were used in the Salmonella mutagenicity assay. Liver S9 fractions from both species resulted in a significantly greater number of His+ revertants (TA98) per plate than observed on the control plates (spontaneous reversion rate). Liver homogenates from adult Sprague-Dawley rats showed a significantly lower potential to activate B(a)P than homogenates from adult BALB/c mice. In both species, male liver microsomal enzymes had a greater potential to activate B(a)P than female microsomal enzymes. These data indicated that in uninduced tissues B(a)P may not be a very powerful mutagenic agent. More detailed in vitro and in vivo studies are needed to assess the precise health risks associated with this environmental pollutant.     

The development of arylsulphatase in the small intestine of the rat

1. Arylsulphatase activity was measured in stomach, proximal and distal third of small intestine, colon, liver and kidney of foetal and neonatal Sprague-Dawley rats and Swiss mice, with nitrocatechol sulphate as substrate. 2. The specific activity in the distal small intestine, but not in the stomach, proximal small intestine or colon, increased about fourfold between 5 and 16 days after birth in both conventional and germ-free rats. 3. No comparable increase occurred in the distal small intestine of the mouse. 4. The specific activity of acid phosphatase in the distal small intestine of the rat rose only slightly when the arylsulphatase activity increased. 5. The pH optimum and Michaelis constant of arylsulphatase activity of the distal small intestine were similar for 1-day-old, 9-day-old and adult rats. 6. When extracts of distal small intestine of 1-day-old and 9-day-old rats were incubated together, the arylsulphatase activities were additive.     

Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels

Dipeptidyl peptidase IV (DPPIV) is a serine peptidase that cleaves N-terminal dipeptides from polypeptides when the second residue is a proline or an alanine. We have recently cloned cDNAs for rat gp110, a membrane glycoprotein with Mr of 110,000 isolated initially from rat liver. Studies reported here establish that the gp110 for which we have cloned cDNAs is DPPIV. Using the antibodies against and cDNA for DPPIV, we have assessed the tissue distribution of DPPIV by molecular approaches. Immunoblot analysis demonstrated that DPPIV is present in the kidney, lung, and small intestine at high levels, in the liver and spleen at moderate levels, and in the heart at low levels. The highest levels of mRNA for DPPIV were detected in the kidney and small intestine as compared to moderate levels found in the lung, liver, and spleen. The lowest levels of DPPIV mRNA were found in the stomach, testis, and heart. No detectable DPPIV protein and mRNA were found in brain or muscle. LDPPIV protein and mRNA are present at much lower levels in fetal livers as compared to the adult liver. Indirect immunofluorescence microscopy demonstrated that DPPIV is localized in the bile canaliculus of hematocytes and in the apical membrane domains of kidney tubule and small intestine. Further studies by Southern blot analysis indicate that DPPIV is encoded by a single gene.     

Renal inactivation of substance P in the rat.

The activity and distribution of substance P-catabolizing enzyme(s) were studied in the rat kidney. Kidney homogenates inactive substance P 5-20 times as fast as do homogenates of intestine, liver, lung, heart or brain. The catabolizing activity was highest in the cortex and decreased progressively down the papilla. Cortex of rat kidney was homogenized and fractions enriched in microsomal membrane, final supernatant, plasma membrane, endoplasmic reticulum, brush border and intact glomeruli were prepared. The identity and homogeneity of the preparations were determined by assaying marker enzymes and by morphological examination. Substance P was catabolized most rapidly by the microsomal and plasma-membrane-enriched fractions, and least rapidly by endoplasmic reticulum or final supernatant fractions. Purified brush border of proximal tubules inactivated substance P more than 10 times as fast as isolated glomeruli. Our experiments show that substance P is catabolized at a rate that is similar to the rates of inactivation of bradykinin and angiotensin II. Further, the distribution of substance P-catabolizing activity in various kidney fractions is similar to the distribution of kininase and angiotensinase activities previously reported.     

Dealkylation of 7-methoxycoumarin as assay for measuring constitutive and phenobarbital-inducible cytochrome P450s

Six alkyl ethers of 7-hydroxycoumarin, ranging from methoxy- to hexoxycoumarin, were studied for their NADPH-dependent metabolism by liver microsomes of male rats treated with phenobarbital (PB) or 3-methyl-cholanthrene (MC). The six alkyl ethers were metabolized by both types of microsomes, forming 7-hydroxycoumarin as the major product. Among the test compounds, 7-methoxycoumarin was unusual in that its dealkylation was inducible only by PB and not by MC. PB increased 7-methoxycoumarin-O-demethylase (MOCD) activity about four- to eightfold. Metyrapone strongly inhibited MOCD in PB-treated microsomes but not in MC-treated microsomes. Similarly, monoclonal antibodies directed toward PB-induced cytochrome P450s selectively suppressed MOCD in PB-treated microsomes. MOCD activity was observed in preparations of SD1 cells containing only cytochrome P450IIB1, while it was not found in preparations of XEM1 cells containing only cytochrome P450IA1. Demethylation of 7-methoxycoumarin was also mediated by the constitutive cytochrome P450 form(s) of liver, lung, small intestine, and kidney (in decreasing order). PB increased MOCD activity of small intestine by 40% but was without effect on the dealkylation activity of lung and kidney. MOCD activity was also detectable in differentiated rat hepatoma lines H4IIEC3 and 2sFou. The studies indicate that dealkylation of 7-methoxycoumarin is a highly sensitive, simple, and practical assay for estimating constitutive and PB-inducible cytochrome P450-dependent monooxygenase activities.     

Age-related changes in mutagen activation by rat tissues

Age-related changes in drug metabolism of the liver, lung and kidney of adult female Long-Evans rats were determined by measuring changes in mutagen formation. Activation of aflatoxin B1 (AFB1), 2-aminofluorene (AF) and 2-acetylaminofluorene (AAF) to mutagenic derivatives was assayed using the Ames Salmonella test system. The promutagens were incubated with tissue fractions from rats ranging in age from 2.5 to 25 months. With all three compounds, hepatic, renal and pulmonary activation was lower in the senescent than in the young adult animals. The largest decrease, however, occurred prior to middle-age, i.e. before 9-13 months. In liver and kidney, little change was detectable between the middle-aged and the old (20-25 months) animals. However, pulmonary metabolism in the oldest animals was slightly higher than in the extracts from the middle-aged rats. The observed decline in mutagen activation may thus be a function of maturation rather than senescence.     

1-Hydroxypyrene as an indicator of the mutagenicity of coal tar after activation with human liver preparations

Liver S9 fractions were prepared from male Wistar rats, either non-induced or induced with Aroclor 1254 and from 5 human kidney transplant donors. The preparations were compared for their ability to metabolize the premutagens present in coal tar to mutagenic metabolites in the Salmonella mutagenicity assay towards strain TA98. Low levels of mutagenicity of coal tar were seen with human S9 preparations. The differences between the S9 mix of the 5 donors in capacity to activate premutagens were approximately 6-fold. The activation of coal tar by rat liver S9 preparations was higher than by the human S9 preparations. The metabolic conversion of pyrene in coal tar to 1-hydroxypyrene by the same human S9 preparations was determined in a parallel assay. 3 human preparations showed a high correlation between the formation of 1-hydroxypyrene and bioactivation of coal tar to mutagenic metabolites. The slope values of the individual regression lines were equal, suggesting that 1-hydroxypyrene is a good indicator for the activation of premutagens present in coal tar.     

Antigenic properties of the loosely bound subnuclear fraction of rat brain

Antibodies against the loosely bound subnuclear fraction (0.35 M NaCl-extractable subnuclear fraction) of rat brain were raised in rabbits, and the distribution of the main antigenic determinants was followed among subcellular fractions of nervous tissue and among homologous nuclear preparations from different tissues. By immunofluorescence a localization restricted to the nucleus was observed, and by microcomplement fixation the antigens appeared to specifically enrich the fraction under examination, being poorly detectable in cytosol, nuclear sap, or deoxyribounucleoproteins of rat brain. No significant cross-reaction was observed by complement fixation with homologous preparations from muscle, liver, kidney, spleen, lung, or thymus of rat, whereas the 0.35 M NaCl-extracted subnuclear fraction from rat testis exhibited an immunoreactivity, although lower than that for brain proteins. After trypsin or ribonuclease treatment, the main antigenic determinants appeared to be protein in nature. The subnuclear fraction under examination, which is believed to be relevant to gene regulation, appears to contain protein antigens mainly concentrated in the nervous system.     

Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells

The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 3-OH-AAF 3-hydroxy-2-acetylaminofluorene - 5/9-OH-AAF a combination of 5 and 9-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8-OH-AAF 8-hydroxy-2-acetylaminofluorene     

Aflatoxin B1 metabolism by 3-methylcholanthrene-induced hamster hepatic cytochrome P-450s

We have studied the activation of aflatoxin B1 by hamster liver microsomes and purified hamster cytochrome P-450 isozymes using a umu mutagen test. The hamster liver microsomes or S-9 fractions were much more active than rat liver microsomes or S-9 fractions in the activation of umu gene expression by aflatoxin B1 metabolites. 3-Methyl-cholanthrene treatment increased aflatoxin B1 activation by hamster liver microsomes. Two major 3-methylcholanthrene-inducible cytochrome P-450 isozymes, P-450 MC1 (IIA) and P-450 MC4 (IA2), were purified from 3-methylcholanthrene-treated hamster liver microsomes, and the metabolism of aflatoxin B1 by these two cytochromes was studied. In the reconstituted enzyme system, both P-450 MC1 and P-450 MC4 were highly active in the activation of aflatoxin B1, and antibodies against these P-450s specifically inhibited these activities. Antibody against P-450 MC1 inhibited the activation of aflatoxin B1 by 20% in the presence of 3-methyl-cholanthrene-treated hamster liver microsomes. In contrast, antibody against P-450 MC4 stimulated the activity by 175%. These results indicated that hamster P-450 MC1 might convert aflatoxin B1 to more toxic metabolite(s), whereas P-450 MC4 might convert aflatoxin B1 to less toxic metabolite(s), than aflatoxin B1 in liver microsomes. The metabolite(s) produced by both hamster cytochrome P-450 MC1 and MC4 were genotoxic in the umu mutagen test.