Tumorigenicity assessments of Per.C6 cells and of an Ad5-vectored HIV-1 vaccine produced on this continuous cell line.

PER.C6, a cell line derived from human embryonic retinal cells transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes, is used to produce E1-deleted Ad5 vectors such as the MRKAd5 HIV-1 gag vaccine. While whole, live PER.C6 cells are capable of growing as tumours when transplanted subcutaneously into immunodeficient nude mice at a high dosage, the process for vaccine production includes filtration steps and other methods which effectively preclude contamination by intact viable substrate cells. However, because of the neoplastic nature of this cell line, we carried out a series of investigations to assess the tumorigenic risk posed by residuals from the cell substrate in a vaccine. To address concerns about transmission of oncogenic DNA, we demonstrated that purified PER.C6 cellular DNA does not induce tumours in newborn hamsters or nude mice. To address concerns about other potential residuals, including hypothetical adventitious tumour viruses, we demonstrated that a PER.C6 cell lysate and a MRKAd5 HIV-1 gag vaccine produced on PER.C6 cells do not induce tumours in newborn hamsters or newborn rats. These results, in conjunction with the wide panel of viral safety tests performed on these cells, support the safety of the PER.C6 as a cell substrate for vaccine production.

Effects of low concentrations of benzene on mouse hematopoietic cells in vivo: a preliminary report.

Evaluation of benzene-induced hematotoxicity following exposure to low concentration is important for understanding mechanisms of toxicity and determining the dose response at benzene levels close to the current occupational exposure limit (1 ppm). Male B6C3F1 mice were exposed to 0, 1, 10, 100, or 200 ppm benzene by inhalation for 6 hr/day, 5 days/week, for 1, 2, 4, or 8 weeks. At each sampling time, we evaluated primitive and committed progenitor cells, differentiating and maturing lineage-specific cells, and stromal cells in the bone marrow; T and B lymphocytes of the spleen and thymus; micronucleated reticulocytes and erythrocytes; and standard blood parameters. At 100 and 200 ppm benzene, there were rapid and significant reductions in number of reticulocytes in the blood, B lymphocytes in the bone marrow and spleen, and an increased frequency of micronucleated reticulocytes in the bone marrow. At 10 ppm, the only parameter affected was a transient reduction in the number of splenic B lymphocytes. There were no significant effects induced by 1 ppm benzene in this study. The present study suggests numbers of B lymphocytes and maturing erythrocytes, and frequency of micronucleated reticulocytes are sensitive indicators of benzene-induced hematotoxicity and will be useful in further investigation of the hematotoxicity induced by 10 to 100 ppm benzene.

Effects of a thirteen-week inhalation exposure to ethyl tertiary butyl ether on fischer-344 rats and CD-1 mice.

The 1990 Clean Air Act Amendments require that oxygenates be added to automotive fuels to reduce emissions of carbon monoxide and hydrocarbons. One potential oxygenate is the aliphatic ether ethyl tertiary butyl ether (ETBE). Our objective was to provide data on the potential toxic effects of ETBE. Male and female Fisher 344 rats and CD-1 mice were exposed to 0 (control), 500, 1750, or 5000 ppm of ETBE for 6 h/day and 5 days/wk over a 13-week period. ETBE exposure had no effect on mortality and body weight with the exception of an increase in body weights of the female rats in the 5000-ppm group. No major changes in clinical pathology parameters were noted for either rats or mice exposed to ETBE for 6 (rats only) or 13 weeks. Liver weights increased with increasing ETBE-exposure concentration for both sexes of rats and mice. Increases in kidney, adrenal, and heart (females only) weights were noted in rats. Degenerative changes in testicular seminiferous tubules were observed in male rats exposed to 1750 and 5000 ppm but were not seen in mice. This testicular lesion has not been reported previously for aliphatic ethers. Increases in the incidence of regenerative foci, rates of renal cell proliferation, and alpha2u-globulin containing protein droplets were noted in the kidneys of all treated male rats. These lesions are associated with the male rat-specific syndrome of alpha2u-globulin nephropathy. Increases in the incidence of centrilobular hepatocyte hypertrophy and rates of hepatocyte cell proliferation were seen in the livers of male and female mice in the 5000-ppm group, consistent with a mitogenic response to ETBE. These two target organs for ETBE toxicity, mouse liver and male rat kidney, have also been reported for methyl tertiary butyl ether and unleaded gasoline.

Genotoxicity and cytotoxicity in male B6C3F1 mice following exposure to mixtures of 1,3-butadiene and styrene.

1,3-Butadiene and styrene are oxidized, in part, by cytochrome P450 2E1 and have been shown to metabolically interact in rodents exposed by inhalation to mixtures of both compounds. Because the reactive metabolites of butadiene and styrene are thought to be responsible for the toxicity of each compound, metabolic interactions may alter the response in animals exposed to mixtures of butadiene and styrene compared with the response in animals exposed to butadiene alone or styrene alone. The purpose of this study was to quantitate alterations in genotoxicity and cytotoxicity in male B6C3F1 mice exposed to mixtures of butadiene and styrene. Male B6C3F1 mice were exposed to 6.25, 62.5, 200, or 625 ppm butadiene alone, 50 ppm styrene alone, or mixtures of 6.25, 62.5, 200, or 625 ppm butadiene and 50 ppm styrene. Genotoxicity was assessed by quantitating the frequency of micronucleated polychromatic erythrocytes in bone marrow. Cytotoxicity was assessed by counting total spleen and thymus cells and by quantitating the frequency of polychromatic erythrocytes in the peripheral blood. Butadiene and mixtures of butadiene and styrene were genotoxic in mice, as shown by a significant increase in the frequency of micronucleated polychromatic erythrocytes. The increased frequency following exposure to mixtures of butadiene and styrene was not significantly different compared with the frequency following exposure to butadiene alone. Styrene and mixtures of butadiene and styrene were cytotoxic in mice, as shown by significantly decreased number of spleen cells. Exposure to mixtures of butadiene and styrene with butadiene concentrations of 62.5 or 625 ppm significantly reduced the number of thymus cells. Exposure to 200 ppm or 625 ppm butadiene alone, or to mixtures of 200 ppm or 625 ppm butadiene and 50 ppm styrene, significantly reduced the frequency of polychromatic erythrocytes in the peripheral blood. The results of the study demonstrate that exposure to mixture of butadiene and styrene does not reduce the respective genotoxicity of butadiene or cytotoxicity of styrene.

Immunotoxicological effects of benzene inhalation in male Sprague-Dawley rats.

The inhalation of benzene is toxic to various components of the immunologic system in rodents. Spleen and thymus weights, total spleen and femur marrow cell counts, enumeration of spleen B- and T-lymphocytes, and an assessment of humoral immunocompetence, were used to evaluate the immunotoxicity of benzene in male Sprague-Dawley rats. Rats were exposed to 0, 30, 200 or 400 ppm benzene for 6 h/day, 5 days/week for 2 or 4 weeks. An early indicator of immunotoxicity was a reduction in the number of B-lymphocytes after 2 weeks of 400 ppm. After 4 weeks of 400 ppm, there was a reduction in thymus weight and spleen B-, CD4+/CD5+ and CD5+ T-lymphocytes. Rats exposed to 30, 200 or 400 ppm benzene for 2 or 4 weeks and challenged with sheep red blood cells developed a humoral response comparable to that of the control (0 ppm) animals. Enumeration of spleen T- and B-lymphocytes in rats exposed to benzene and challenged with SRBC showed only a transient reduction in spleen B-lymphocytes after 2 weeks of exposure to 400 ppm. These data suggest that there are no immunotoxicological effects of exposure to 200 ppm benzene or less, in rats exposed for 6 h/day, 5 days/week for 2 or 4 weeks.

Detection of beryllium sensitivity using a flow cytometric lymphocyte proliferation test: the Immuno-Be-LPT.

Measurement of lymphocyte proliferation to detect hypersensitivity to beryllium (Be-LPT) in vitro is done presently using a method based on tritiated thymidine incorporation. Although this method is sensitive it gives no information on cell viability or responding lymphocyte subsets. We have developed reliable and simple flow cytometric assays for lymphocyte proliferation testing (Immuno-Be-LPT) by combining immunophenotyping with bromodeoxyuridine (BrdU) incorporation or DNA content using propidium iodide (PI) or 4'6'-diimidazolin-2-phenylindole (DAPI). Evaluation of beryllium-induced lymphocyte proliferation in blood cells from seven patients with chronic beryllium disease (CBD) and 120 beryllium workers by both the Bc-LPT and the Immuno-Be-LPT showed agreement between the tests. The Immuno-Bc-LPT provided additional information about the specific type of lymphocytes responding. CD4+ lymphocytes proliferated in response to beryllium in blood samples from all seven CBD individuals and CD8+ lymphocytes proliferated in six of the seven. Four beryllium workers without CBD had positive responses to beryllium primarily in the CD8+ cells. The use of the individual's own plasma supported a greater beryllium or tetanus-induced proliferation of CD4+ lymphocytes when compared to commercial human serum. The response of CD4+ lymphocytes measured in the Immuno-Be-LPT may provide a new marker for the diagnosis of CBD.

Beryllium sensitivity is linked to HLA-DP genotype.

Chronic beryllium disease (CBD) appears to arise from a combination of both exposure and genetic risk factors. A distinguishing feature of CBD is beryllium hypersensitivity, which can be measured in vitro by a lymphocyte proliferation test. The objective of this study was to determine whether certain allelic variations of the HLA-DPB1 gene, which had been observed previously in CBD, could be found in a group of individuals having beryllium hypersensitivity, but no symptoms of CBD. A flow cytometry-based Lymphocyte Proliferation Test combined with immunophenotyping (Immuno-LPT) was used to detect CD4+ and CD8+ T cell proliferation in response to in vitro stimulation with beryllium. The HLA-DPB1 haplotypes of the same individuals were determined by automated DNA sequencing. Twenty-two out of 25 beryllium-sensitive, non-CBD individuals were found to be carriers of the HLA-DPB1 gene having a substitution of a glutamic acid at position 69 in Exon 2 (Glu69), and a significantly high percentage (24%) were Glu69 homozygotes. Most of the CD4+ responders on the Immuno-LPT (10/14) carried rare, non-*0201 Glu69 DPB1 alleles; while most of the non-CD4+ responders (9/11) were common Glu69 carriers (*0201 or *0202) or non-Glu69 individuals (non-Glu69/non-Glu69). This is the first direct evidence that HLA-DP genotype is linked to a phenotypic response that occurs in beryllium sensitization in the absence of clinical CBD.

Effects of benzene on splenic, thymic, and femoral lymphocytes in mice.

Chronic exposure to high concentrations of benzene, primarily by inhalation, can affect the function of the human immune system. Limited data are available on the immunotoxic effects of low concentrations of benzene. This study evaluated the effects of 1, 5, 10, 100, and 200 ppm benzene on lymphocytes in mice exposed by inhalation for up to 8 weeks. Exposure to 100 or 200 ppm benzene induced rapid and persistent reductions in femoral B-, splenic T- and B-, and thymic T-lymphocytes. The percentage of femoral B-lymphocytes and thymic T-lymphocytes in apoptosis was increased 6- to 15-fold by 200 ppm benzene compared to controls. Replication of femoral B-lymphocytes was increased during the exposure period in the bone marrow as a compensation for the lymphocyte loss induced by 100 and 200 ppm benzene. Exposure of mice to 10 ppm benzene or less did not have a statistically significant effect on numbers or replication of the lymphocyte populations evaluated. A reduced number of splenic B-lymphocytes after 2 weeks of exposure to benzene appeared to be the most sensitive end point and time point for evaluating benzene cytotoxicity in this study.

Inhibition of myelopoiesis by conditioned medium from cultured canine thymic cells exposed to estrogen.

Therapeutic doses of estrogens can cause fatal bone marrow damage in dogs, which are more sensitive than other species to these myelotoxic effects. Investigations with mice indicated that estrogens did not directly damage the bone marrow progenitor cells, but that the thymus responded to estrogen by producing a factor that inhibited bone marrow granulocyte-macrophage progenitor cell replication. A similar estrogen-induced myelopoiesis-inhibitory factor was produced by canine thymic cells in culture. This canine factor was more inhibitory to myelopoiesis than was the murine factor. Canine bone marrow progenitor cell growth was not significantly inhibited by direct estrogen treatment, which supported evidence for indirect thymus mediation of estrogen toxicity in vivo. Estrogen receptors were detected in canine nonlymphoid thymic cells by use of immunocytochemical staining. These findings indicate that the high estrogen sensitivity of dogs may relate to greater estrogen-induced myelopoiesis-inhibition by the thymus.

Inhibition of myelopoiesis by serum from dogs exposed to estrogen.

The mechanism of estrogen-induced myelotoxicosis is unknown, although evidence indicates that estrogen does not directly damage the bone marrow granulocyte-macrophage progenitor cells and that the thymus is a probable mediator of the bone marrow suppression. Estrogen-induced production of a myelopoiesis-inhibitory factor by canine thymic stromal cells in vitro has been observed. Then, presence of a myelopoiesis-inhibitory factor in canine serum was investigated immediately after estrogen administration in vivo. Maximal reduction in colony-forming units-granulocyte/macrophage growth by sera from individual dogs varied. Individual dog sensitivity to estrogen-induced myelotoxicosis is seen clinically, and the cause is unknown. This serum factor could have a role in the eventual bone marrow hypoplasia seen in estrogen-treated dogs and is possibly the same factor produced by cultured thymic stromal cells exposed to estrogen.

Carcinogenicity of inhaled benzene in CBA mice.

This study investigated benzene-induced neoplasia in CBA/Ca mice, with special emphasis on hematopoietic tissues. Ten-week-old male CBA/Ca mice were exposed to 300 ppm benzene via inhalation for 6 hr/day, 5 days/week, for 16 weeks and held 18 months after the last exposure. There were 125 benzene-exposed and 125 sham-exposed mice. Malignant lymphoma was a statistically significant cause of early mortality in the benzene-exposed mice. Fourteen benzene-exposed mice developed lymphoma (lymphoblastic, lymphocytic, or mixed) as compared to only 2 sham-exposed mice. Benzene-exposed mice also developed preputial gland squamous cell carcinomas (60% in benzene-exposed vs 0% in sham-exposed) and had an increased incidence of lung adenomas (36% vs 14%). Moderate to marked granulocytic hyperplasia was present in benzene-exposed animals, with a 36% incidence in the bone marrow and 6% in the spleen, as compared to the sham-exposed with 8 and 0%, respectively. Interpretation of the granulocytic response as a direct effect of benzene was complicated by the presence of inflammation in the mice. Although inhaled benzene was clearly carcinogenic in CBA mice, it did not induce granulocytic leukemia.

Benzene-induced micronuclei in erythrocytes: an inhalation concentration-response study in B6C3F1 mice.

High concentrations (300-1000 p.p.m.) of benzene have been shown to induce an increase in the frequency of micronucleated erythrocytes in mice. This study investigated the mutagenicity of benzene at lower concentrations, including the current limit for occupational exposure, 1 p.p.m. The frequencies of micronucleated polychromatic erythrocytes (MPCE) in the bone marrow and blood and micronucleated normochromatic erythrocytes (MNCE) in the blood of male B6C3F1 mice were measured following inhalation of benzene at 0, 1, 10, 100 or 200 p.p.m. during an 8 week exposure period. Only 100 and 200 p.p.m. benzene induced a statistically significant increased frequency of micronucleated erythrocytes in the bone marrow and blood. The frequency of MPCE plateaued at week 2 with 43/1000 (100 p.p.m.) and 86/1000 (200 p.p.m.) in the bone marrow as compared with 10/1000 for controls. The frequency of MNCE in the blood progressively increased to 13.4/1000 (100 p.p.m.) and 32.5/1000 (200 p.p.m.) at week 8 as compared with 1.8/1000 for controls. Cytotoxicity of replicating and maturing erythrocytes by 100 and 200 p.p.m. benzene delayed the accumulation of MNCE in the blood. There was not a statistically significant increase in the frequency of micronucleated erythrocytes, as an indicator of mutagenicity, with inhalation of 1 or 10 p.p.m. benzene over an 8 week period. A quadratic curve fit the bone marrow MPCE data of mice exposed to up to 200 p.p.m. benzene with a high correlation (R2 = 0.94) and could not be rejected based on lack of fit.

Pharmacokinetics of inhaled [14C]methanol and methanol-derived [14C]formate in normal and folate-deficient cynomolgus monkeys.

Large-scale use of methanol (MeOH) as an automotive fuel may increase exposure of the public to MeOH vapor, necessitating the need for additional data for an adequate human health risk assessment. Formate is accepted as the toxic metabolite of MeOH, its metabolism is folate-dependent, and potentially sensitive folate-deficient subpopulations (e.g., pregnant women) exist that may be at higher risk to low-level methanol exposure. This study determined the pharmacokinetics of [14C]MeOH and [14C]formate in normal and folate-deficient (FD) monkeys following inhalation of environmentally relevant concentrations of [14C]MeOH. Four normal adult female cynomolgus monkeys were anesthetized (isoflurane) and exposed by lung-only inhalation to 10, 45, 200, and 900 ppm [14C]MeOH for 2 hr. Monkeys were then placed on a FD diet until folate concentrations consistent with moderate deficiency (29-107 ng/ml) developed in red blood cells and then reexposed to 900 ppm (900-FD) for 2 hr. Average (+/- SD) end-of-exposure blood [14C]MeOH concentrations were 0.65 +/- 0.3, 3.0 +/- 0.8, 21 +/- 16, 106 +/- 84, and 211 +/- 71 microM, while average (+/- SD) peak blood [14C]formate concentrations were 0.07 +/- 0.02, 0.25 +/- 0.09, 2.3 +/- 2.9, 2.8 +/- 1.7, and 9.5 +/- 4.7 microM following MeOH inhalation at 10, 45, 200, 900, and 900-FD ppm, respectively. The blood concentration of [14C]MeOH-derived formate from all exposures was 10 to 1000 times lower than the endogenous blood formate concentration (0.1 to 0.2 mM) reported for monkeys. These results suggest that low-level exposure to MeOH would not result in elevated blood formate concentrations in humans under short-term exposure conditions.

Benzene-induced hematotoxicity and bone marrow compensation in B6C3F1 mice.

Long-term inhalation exposure of benzene has been shown to cause hematotoxicity and an increased incidence of acute myelogenous leukemia in humans. The progression of benzene-induced hematotoxicity and the features of the toxicity that may play a major role in the leukemogenesis are not known. We report the hematological consequences of benzene inhalation in B6C3F1 mice exposed to 1, 5, 10, 100, and 200 ppm benzene for 6 hr/day, 5 days/week for 1, 2, 4, or 8 weeks and a recovery group. There were no significant effects on hematopoietic parameters from exposure to 10 ppm benzene or less. Exposure of mice to 100 and 200 ppm benzene reduced the number of total bone marrow cells, progenitor cells, differentiating hematopoietic cells, and most blood parameters. Replication of primitive progenitor cells in the bone marrow was increased during the exposure period as a compensation for the cytotoxicity induced by 100 and 200 ppm benzene. In mice exposed to 200 ppm benzene, the primitive progenitor cells maintained an increased percentage of cells in S-phase through 25 days of recovery compared with controls. The increased replication of primitive progenitor cells in concert with the reported genotoxicity induced by benzene provides the components necessary for producing an increased incidence of lymphoma in mice. Furthermore, we propose this mode of action as a biologically plausible mechanism for benzene-induced leukemia in humans exposed to high concentrations of benzene.