Absorption, distribution, and excretion of ammonium perfluorooctanoate (APFO) after oral administration to various species.

Male and female mice, rats, hamsters, and rabbits were treated with a single oral dose of 14C-ammonium perfluorooctanoate (APFO), and the excretion and tissue distributions were followed for 120 h (168 h in the rabbit). Substantial sex and species differences in the excretion and disposition of 14C-radioactivity derived from 14C-labeled APFO were observed in this study. The female rat and the male hamster excreted more than 99% of the original 14C activity by 120 h after dosing; conversely, the male rat and the female hamster excreted only 39% and 60% of the original 14C activity, respectively, by 120 h postdosing. The male and female rabbits excreted the 14C activity as rapidly and completely as the female rat and the male hamster, whereas male and female mice excreted only 21% of the original 14C activity by 120 h postdosing. The rapid excretors (female rat, male hamster, and male and female rabbits) contained negligible amounts of 14C in organs and tissues at sacrifice. The slow excretors exhibited the highest 14C concentrations in the blood and liver followed by the kidneys, lungs, and skin.

Aneuploidy: a report of an ECETOC task force.

Aneuploidy plays a significant role in adverse human health conditions including birth defects, pregnancy wastage and cancer. Although there is clear evidence of chemically induced aneuploidy in experimental systems, to date there are insufficient data to determine with certainty if chemically induced aneuploidy contributes to human disease. However, since there is no reason to assume that chemically induced aneuploidy will not occur in human beings, it is prudent to address the aneugenic potential of chemicals in the safety assessment process. A wide range of methods has been described for the detection of chemically induced aneuploidy including subcellular systems, tests with fungi, plants and Drosophila as well as in vitro mammalian systems and in vivo mammalian somatic and germ cell assays. However, none of these methods is sufficiently validated or widely used in routine screening. Underlying the efforts to develop aneuploidy-specific assays is the presumption that current genetic toxicology tests do not detected chemicals that have aneuploidy-inducing potential. To address this, we have critically evaluated data from standard genetic toxicology assays for 16 known or suspected aneugens. The conclusions from the review are listed below. 1. At present there are only nine chemicals that can be classified as definitive aneugens, as determined by positive results in in vivo rodent assays. 2. As expected, the majority of definitive and suspected aneugens are negative in the bacterial mutation assay. 3. The majority of definitive aneugens evaluated induce polyploidy in vitro. With few exception, they also induced structural chromosome aberrations in vitro. 4. All of the definitive aneugens that have been sufficiently tested induce micronuclei in rodent bone marrow cells in vivo. A number of these chemicals also induced structural chromosome aberrations in vivo. 5. There is no evidence for a unique germ cell aneugen, that is a chemical that induces aneuploidy in germ cells and not in somatic cells. Furthermore, an analysis of several databases indicates the proportion of chemicals which induce polyploidy and not chromosome aberrations in vitro is low. Based on these conclusions, the following recommendations are made: for screening purposes, a standard genotoxicity test battery (including an in vitro cytogenetic assay with an assessment of polyploidy and clastogenicity at the same harvest time) should be performed; in the absence of polyploidy induction in vitro no further evaluation of aneuploidy-inducing potential is needed; if polyploidy is observed, in vitro follow-up testing to investigate further the aneuploidy-inducing potential should be conducted; such follow-up testing will generally start with the conduct of a standard in vivo somatic cell micronucleus assay; if the in vivo somatic cell micronucleus assay is negative, with adequate evidence of exposure of the bone marrow to the test compound, no further testing of aneuploidy-inducing potential is needed; if the in vivo somatic cell micronucleus assay is positive, further information on mechanisms of micronucleus induction can be obtained by using kinetochore/centromeric staining in vitro and/or in vivo; an assessment of potential germ cell aneuploidy activity may then be considered; aneuploidy induction which does not involve the direct interaction of a chemical or its metabolite(s) with DNA is expected to have a threshold. This must be considered in the risk assessment of such chemicals; this is not addressed by current risk assessment guidelines.

Evaluation of hexamethylphosphoramide for gene mutations in Salmonella typhimurium using plate incorporation, preincubation, and suspension assays.

Hexamethylphosphoramide (HMPA), a potent rat nasal carcinogen by inhalation, and three of its metabolites, pentamethylphosphoramide (PMPA), trimethylphosphoramide (TriMPA), and formaldehyde (HCHO), were assessed in Salmonella typhimurium gene mutation assays using various protocols, including plate incorporation, preincubation and suspension assays. HMPA (tested up to 15,000 micrograms/plate) was not mutagenic in plate incorporation or preincubation assays with or without metabolic activation. HCHO was mutagenic in the plate incorporation and preincubation assays (tested up to 150 micrograms/plate). In suspension assays, however, HMPA (tested up to 40 mg/ml), PMPA (up to 44 mg/ml) and HCHO (up to 45 micrograms/ml), but not TriMPA (up to 29 mg/ml), were mutagenic. HMPA and PMPA were positive only with activation. HMPA's mutagenicity was optimized using a relatively high level of rat liver S9 protein (3.5 mg/plate) in the metabolic activation mixture. Semicarbazide, an HCHO trapping agent, added at concentrations up to 167 micrograms/ml, markedly inhibited the mutagenic activities of HMPA and PMPA suggesting that HCHO generation may play a role in their mutagenicity. These studies show that HMPA is mutagenic in a modified Salmonella typhimurium reverse mutation assay with metabolic activation. Successive N-demethylation of HMPA eventually eliminates the mutagenic activity which further suggests that HMPA's mutagenic activity is related to the release of HCHO.

Evaluation of benomyl and carbendazim in the in vivo aneuploidy/micronucleus assay in BDF1 mouse bone marrow.

Benomyl and its active metabolite carbendazim were investigated in BDF1 mouse bone marrow to establish whether micronuclei induced by these fungicides are caused by clastogenic or aneugenic events. Micronuclei were evaluated for kinetochores using immunofluorescent antikinetochore antibodies. Kinetochore positive (K+) micronuclei are likely to arise from chromosome loss since they presumably contain intact kinetochores and are indicative of aneuploidy. Conversely, kinetochore negative (K-) micronuclei are mostly likely to contain acentric chromosome fragments arising primarily from clastogenic damage. Benomyl and carbendazim were administered as single oral doses of 0.3, 8.6 or 17.2 mmol/kg (for benomyl, equivalent to 100, 2500 or 5000 mg/kg; for carbendazim, equivalent to 66, 1646 or 3293 mg/kg). Both compounds were positive in the micronucleus test at doses of 8.6 and 17.2 mmol/kg, and an average of 82% (benomyl) and 87% (carbendazim) of the total micronucleated polychromatic erythrocytes were K+. No effects were seen with either fungicide at 0.3 mmol/kg. These results are analogous to findings with known aneugens such as vincristine but are in contrast to results with classical clastogens such as cyclophosphamide. Thus, benomyl and carbendazim induce micronuclei in mouse bone marrow cells primarily through an aneugenic mechanism.

Evaluation of carbendazim for gene mutations in the Salmonella/Ames plate-incorporation assay: the role of aminophenazine impurities.

Benomyl (methyl [1-[(butylamino)carbonyl]-1H-benzimidazol-2- yl]carbamate) and its major metabolite carbendazim (methyl 2-benzimidazolecarbamate) are major agricultural systemic fungicides. These compounds inhibit fungal microtubular function and thereby cause nondisjunction of chromosomes at cell division. Several investigators have proposed that these compounds can also cause gene mutations (base-pair substitutions). In this laboratory, no mutagenic activity was observed with either benomyl (analytical grade) or Benlate (samples tested up to 500 and 1200 micrograms/plate, respectively, the limit of cytotoxicity) in the Salmonella/Ames plate-incorporation test in either base-pair substitution (TA100 and TA1535) or frameshift-sensitive (TA98 and TA1537) strains with or without S9 metabolic activation. However, some carbendazim preparations caused mutations in frameshift-sensitive strains at very high concentrations (> or = 5000 micrograms/plate) with metabolic activation. The mutagenic activity was not due to the major carbendazim metabolite, methyl (5-hydroxy-1H-benzimidazol-2-yl)carbamate (5-OH MBC), since 5-OH MBC was not mutagenic with (up to 20,000 micrograms/plate) or without (up to 16,000 micrograms/plate) activation. Subsequently, two highly mutagenic contaminants, 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) were detected in mutagenic carbendazim samples. In those samples, DAP and AHP contaminant levels ranged as high as 46.5 and 11.6 ppm, respectively. No evidence of mutagenicity could be detected in preparations in which the DAP content was < 1.8 ppm. The mutagenic activity of these two contaminants was further investigated in strain TA98. Without activation, DAP and AHP were positive at test concentrations as low as 5 and 10 micrograms/plate, respectively. In the presence of S9, mutations were detected at much lower concentrations (beginning at 0.025 and 0.05 microgram/plate, respectively). These results indicate that carbendazim samples containing DAP or AHP at levels as low as 5 or 10 ppm, respectively, would be positive in the Salmonella/Ames test with activation when tested at 5000 micrograms/plate. Purified carbendazim is not mutagenic.

Assessing the risk of heritable gene mutation in mammals: drosophila sex-linked recessive lethal test and tests measuring DNA damage and repair in mammalian germ cells.

The former U.S. EPA OPPT tiered test scheme for heritable gene mutations included the Drosophila sex-linked recessive lethal (SLRL) test in which positive results triggered the mouse specific locus (MSL) test. However, review of available literature indicated that the evaluation of mutations in the germ cells of this insect is not a good predictor of the risk of heritable gene mutations in mammals. The database contained 29 compounds for which there were conclusive MSL test results in either spermatogonial and/or postspermatogonial cells. Results in the SLRL test were available for 27 of those compounds. Of the 24 SLRL-positive chemicals, only 13 (54%) induced heritable mutations in mice; the three SLRL-negative compounds were nonmutagenic in mouse germ cells. The overall concordance between the two tests was 59%. In contrast, results of unscheduled DNA synthesis (UDS: 18 chemicals) and alkaline elution (AE: 14 chemicals) assays in rodent testicular cells following in vivo exposure correlated well with results in the MSL test (83% and 86%, respectively). MSL test results in spermatogonia and postspermatogonia were also compared separately to the SLRL, UDS, and AE assays. The concordances for the two cell types in the SLRL relative to the MSL test were 36% and 79%, respectively, indicating that the SLRL test is extremely poor in predicting heritable gene mutations in mammalian spermatogonia. Concordances for UDS and AE assays relative to MSL test results in spermatogonia (53% and 54%, respectively) and postspermatogonia (91% and 100%, respectively) were greater. Based on these analyses, the U.S. EPA OPPT has revised its tiered test scheme using assays for interaction with gonadal DNA (e.g., UDS and AE) in place of the SLRL test.

In vitro and in vivo genotoxicity of 1,3-butadiene and metabolites.

1,3-Butadiene and two major genotoxic metabolites 3,4-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB) were used as model compounds to determine if genetic toxicity findings in animal and human cells can aid in extrapolating animal toxicity data to man. Sister chromatid exchange (SCE) and micronucleus induction results indicated 1,3-butadiene was genotoxic in the bone marrow of the mouse but not the rat. This paralleled the chronic bioassays which showed mice to be more susceptible than rats to 1,3-butadiene carcinogenicity. However, 1,3-butadiene did not induce unscheduled DNA synthesis (UDS) in the rat or mouse hepatocytes following in vivo exposure. Likewise, UDS in rat and mouse hepatocytes in vitro was not induced by EB or DEB. Salmonella typhimurium gene mutation (Ames) tests of 1,3-butadiene using strains TA1535, TA97, TA98, and TA100 and employing rat, mouse, and human liver S9 metabolic systems were barely 2-fold above background only in strain TA1535 at 30% 1,3-butadiene in air with induced and uninduced rat S9 and mouse S9 (uninduced). 1,3-Butadiene was negative in in vitro SCE studies in human whole blood lymphocytes cultures treated in the presence of rat, mouse, or human liver S9 metabolic activation. In general, 1,3-butadiene is genotoxic in vivo but is a weak in vitro genotoxin.

In vivo sister chromatid exchange and micronucleus induction studies with 1,3-butadiene in B6C3F1 mice and Sprague-Dawley rats.

Male B6C3F1 mice and Sprague-Dawley rats were exposed for 2 days, 6 h/day to 1,3-butadiene (BD) by inhalation (nose only) and their bone marrow cells were evaluated for the induction of micronuclei (MN) and sister chromatid exchanges (SCEs). A significant dose-dependent increase in MN induction was observed in mice. At 100 p.p.m., the frequency of micronucleated polychromatic erythrocytes was 6-fold above control with a maximal induction of 38-fold at 10,000 p.p.m. A significant increase in SCEs was also observed in mouse bone marrow cells starting at 100 p.p.m. with a 4-fold increase over the control evident at 10,000 p.p.m. The highest tested no observed effect level for both endpoints was 50 p.p.m. In contrast, rat bone marrow cells did not exhibit significant increases in micronucleated polychromatic erythrocytes or SCEs. These results indicate that BD is genotoxic in the bone marrow of the mouse but not the rat. This paralleled the chronic bioassays which showed mice to be more susceptible than rats to BD carcinogenicity.

Involvement of deoxyribonuclease activity in the differential sedimentation rates of nucleoids from non-transformed and transformed mouse embryo fibroblasts.

Lysis of non-transformed confluent C3H10T1/2C18 mouse embryo fibroblasts in the presence of detergents, high concentrations of salt and EDTA on top of neutral sucrose gradients revealed a reduced sedimentation rate of the resulting nucleoids from these cells compared to those from exponentially growing non-transformed cells or from transformed cells. Exposure of confluent cells to 1000 rads of X-ray had no effect on this rate of nucleoid sedimentation; and ethidium bromide titration and alkaline sucrose analysis suggested the presence of discontinuities in the DNA. An endonucleolytic activity could be extracted from nuclei of these cells with 0.5 M NaCl, indicating a very tight association with the chromatin. Such an enzyme in non-transformed confluent cells may account for the differences in nucleoid structure and may be related to changes in cell function with normal arrest of cell growth. There was no growth-phase effect on the properties of nucleoids from transformed cells.

Enhancement of growth of human colon tumor cell lines by feeder layers of murine fibroblasts.

The effects of feeder layers of C3H/10T 1/2 cells on the growth of human and mouse cell lines were tested. Feeder layers increased colony formation by cultured cancerous cells in semisolid medium over controls grown in semisolid medium without feeder layers. Maximal colony formation was also attained at a faster rate when feeder layers were used. Plating efficiency by cancerous cells obtained directly from xenografts was increased threefold to fivefold in tissue culture when feeder cells were present as confluent monolayers.

Evidence that estrogen-receptor-negative, progesterone-receptor-positive breast and ovarian carcinomas contain estrogen receptor.

A small number of primary metastatic breast carcinomas are estrogen-receptor-negative and progesterone-receptor-positive (ER-, PGR+) under the normal ligand-binding assay or sucrose density gradient conditions. Among more than 500 tumors analyzed in this laboratory over a year and a half, 28 cases fit this category, 18 of which were patients 51 years of age or younger (Group A) and 7 were patients over the age of 56 (Group B), The ages of three patients were unknown (Group C). By treatment of each of those tumor cytosols with dextran-coated charcoal before the assay was done, 13 of group A became positive (ER range 10-87 fmol/mg protein); 1 was borderline (ER 3-9 fmol/mg protein); 1 became positive only on sucrose gradient determination, and 2 remained negative. In comparison, two patients in group B shifted from borderline ER to ER+ and only one ER- became ER+ at 10 fmol/mg protein. The data provide additional rationale for determining both ER and PGR in all patients, and have obvious implications for the need of standard methods of determining ER and PGR in the prognosis of women with breast cancer.

The importance of short term exposure of C3H 10T1/2 cells to polycyclic hydrocarbons: evidence for hydrocarbon-mediated anticarcinogenic activity.

Since it has been shown that transformation frequencies (TF) of cultured mammalian cells exposed to polycyclic aromatic hydrocarbons (PAH) reach a maximum with increasing PAH concentration and then decline, we have examined TF in C3H 10T1/2 CL8 (10T1/2) cells as a function of an additional parameter of treatment, length of exposure. A 15-min exposure to either benzo[alpha]pyrene (BP) or 3-methylcholanthrene (3-MC), at concentrations ranging from 0.3 to 10 micrograms/ml, was sufficient to induce transformation suggesting that in 10T1/2 cells, non-induced enzymes of the cytochrome P-450 system are involved in the metabolic activation of PAH. At lower BP concentrations (0.3-1.25 micrograms/ml), TF generally increased with exposure time; at higher BP concentrations (2.5-10 micrograms/ml) maximal TF were achieved with 3 h of exposure. For 3-MC, maximal TF occurred at 0.5-1 h with a concentration of 10 micrograms/ml and at 1-6 h with lower concentrations. Moreover, low TF were obtained after 12-h and 24-h exposures to 10 micrograms/ml BP or 3-MC. These results show that TF depend on both the length of exposure and concentration of PAH. Since both BP and 3-MC are extensively metabolized to polyoxygenated derivatives and conjugates, we suggest that certain metabolites may be anticarcinogenic or antipromoting agents. The identities of such metabolites are yet to be determined.

An analysis of clinical and laboratory features of acute lymphocytic leukemias with emphasis on 35 children with pre-B leukemia.

In 35 of 191 patients with acute lymphocytic leukemia (ALL) malignant cells were similar in phenotype to B-lymphocyte precursors. Both these patients' lymphoblasts and normal pre-B-cells contain cytoplasmic immunoglobulin (Ig) mu heavy chains, but have no surface Ig. In patients with pre-B leukemias, lymphoblasts containing cytoplasmic mu chains alone were often accompanied by cells of identical morphology that expressed no Ig and less frequently by lymphoblasts bearing scant amounts of surface mu. This spectrum of cellular Ig expression suggests that "null," pre-B, and intermediate pre-B/B ALLs represent closely related malignancies with complete or partial arrests at different stages of maturation. When pre-B, B, T, and "null" cell categories of ALL were compared for 22 different clinical and laboratory features, including remission rate and short-term remission duration, no statistical differences were observed between the pre-B and "null" groups. These early results suggest that pre-B-cell leukemias represents a relatively good prognostic subclass of ALL, do not require more intensive treatment than that proven to be effective for "null" cell ALL, and should be distinguished from the less common, but more clinically aggressive, B-cell subclass of ALL. Longer follow-up will be required to confirm these preliminary conclusions.

Comparison of steroid receptor levels in renal-cell carcinoma and autologous normal kidney.

Since a number of renal-cell carcinomas regress with hormonal manipulation, we have identified and measured the levels of estrogen, progestin and glucocorticoid receptors in 47 autologous pairs of normal and neoplastic kidney tissues. High-affinity receptors for these hormones were detected in kidney tissues of both sexes by means of a dextran-coated charcoal assay. Glucocorticoid receptors were demonstrated in renal cancer tissues for the first time, and were higher in the tumor (mean 31.3 +/- SEM 5.6) than in the normal tissue (mean 18.5 +/- 3.1 fmol/mg cytosol protein). There was a significant difference in the quantities of progestin receptors (expressed as fmol/mg cytosol protein) in normal (mean 18.4 +/- SEM 3.3) versus neoplastic (mean 10.4 +/- SEM 4.0) kidney specimens (p less than 0.007). There was a significant difference between the binding affinity of the progestin receptor in the male tumors (Kd = 2.2 +/- SEM 0.9 nM, n = 10) and that of the females, (Kd = 9.3 +/- SEM 6.5 nM) (p less than 0.04). When an affinity of less than 9.9 X 10(-9) M and greater than 10 fmol/mg cytosol protein were used as criteria for classifying a tissue as positive for progestin receptors, only 17% of tumors contained these receptors while 45% of normal tissues exhibited them. According to these criteria, no differences were observed in the frequency of occurrence of either estrogen receptors or glucocorticoid receptors in tumor versus normal kidney. Data from this study suggest that the use of endocrine therapy should be re-examined in the treatment of renal-cell carcinoma.

Activity and kinetic properties of basal aryl hydrocarbon hydroxylase during proliferation in the transformable C3H 10T1/2 CL8 cell line.

Basal aryl hydrocarbon hydroxylase (AHH) activity and its kinetic properties were studied as a function of proliferation in C3H mouse embryo 10T1/2 CL8 cells. Activity was low in freshly plated cells, increased during exponential growth, peaked at confluency, and then declined. The apparent Km-values for benzo[a]pyrene (BP) and NADPH were less in proliferating (approx. 0.37 microM BP, 3.3 nM NADPH) than in confluent cells (0.74-1.39 microM BP, 33.4-53.4 nM NADPH). Cells at different growth states responded differently to benz[a]anthracene (BA) and aminophylline, an inhibitor of cyclic nucleotide phosphodiesterases. When cells were harvested at the mid log phase of growth, 12 h of exposure to aminophylline caused maximum induction, while 24 h of BA treatment were required. In contrast, at early confluence, 12 h of BA treatment gave the greatest levels of activity, while exposure to aminophylline did not induce AHH. In fact, decreases in activity were observed. These differences are indicative of different regulatory mechanisms for BA and aminophylline induction. They also suggest the regulation of basal AHH by cyclic nucleotides changes during growth. The exposure times giving maximum activity were used to determine the kinetic properties of BA-induced activity. As with basal AHH, the Km-value for BP was less in log phase (0.2-0.4 microM BP) than in confluent cells (0.64-1.05 microM BP). Moreover, the Km-values for BP and NADPH in control cultures at confluency (0.10-0.14 microM BP, 15.4-23.2 nM NADPH) were less than those for BA-treated cells (0.64 microM BP, 37.9-54.8 nM NADPH) under the same nutritional conditions. The finding that the Km-values for BP is lower in rapidly dividing cells than in confluent cells may help to explain why proliferating cells are more susceptible to transforming agents.

Establishment of mouse colonic carcinoma cell lines with different metastatic properties.

Tumorigenic cell lines were established in culture from three transplantable mouse colonic carcinomas designated CT 26, CT36, and CT 51. The cultured lines were characterized for the retention of the biological characteristics of the parental lines. All three cultured lines retained the ability to form tumors in vivo. Serially transplanted parental lines CT 26 and CT 51 grew at a faster rate than did CT 36 and showed a greater propensity for the formation of lung metastases. Similar characteristics were exhibited by the tumors formed from the injection of cultured cells. The cultured cell lines were also evaluated with respect to a number of in vitro markers for cancer. Cultured CT 26 and CT 51 cells formed tumors at lower inocula than did CT 36. CT 26 and CT 51 showed anchorage-independent growth and lack of contact inhibition, while CT 36 grew as a strict monolayer and did not form colonies in 0.27% agarose. CT 26 had the highest saturation density of the cell lines when grown in media supplemented with either 10 or 2.5% fetal bovine serum, while CT 51 had the lowest saturation density under these conditions. The varying degrees of malignancy exhibited by the three cell lines and the overall retention of the biological characteristics of the parental lines by the cultured lines suggest that the cultured cells (without the contaminating stromal elements present in the serially transplanted lines) will provide suitable material for the investigation of the molecular bases of these malignant characteristics.

Mutations and cell transformation with 2-azido-9-fluorenone oxime.

When photolyzed in situ for as little as 15 s 2-azido-9-fluorenone oxime causes Type II and Type III transformation in C3H 10T 1/2 CL8 cells. In the yeast strain Saccharomyces cerevisiae D-7, simple reversions and gene conversions occurred and also mitotic crossing over, to a lesser extent, but no mitochondril "petite" mutants occurred. No mutations or transformations were induced in the dark or by the light itself.