Genotoxicity testing of an aqueous mistletoe extract in vitro.

An aqueous mistletoe extract (AME, the active principle of Lektinol) was investigated for its genotoxic potential in a wide range of in vitro studies, according to the actual international standard guidelines. Gene mutation tests on bacteria and mammalian cells in vitro all gave negative results. From the findings of cytogenetic studies, there was no indication of a clastogenic activity in human lymphocytes. A cell transformation assay revealed no malignant changes of Syrian hamster embryo cell cultures. In summary, the results suggest that AME has no genotoxic potential in vitro.

A comparison of methods for the estimation of retroviral burden.

The presence of retroviral contamination is of vital concern in the manufacture of cell culture-derived biopharmaceuticals. These cell lines usually have A- or C-type retrovirus-like particles which are visible by transmission electron microscopy (TEM) even when infectivity (IF) or reverse transcriptase activity (RTA) cannot be demonstrated. The supernatant of the post-production cell cultures, therefore, also needs to be evaluated by TEM for viral burden. A major question, however, is how to establish a quantitative viral load estimate for the evaluation of a purification process. The FDA recommends that a purification process for viral contaminants remove or inactivate 3-5 logs over the estimated viral burden. Viral particles are difficult to identify and quantify, however, by conventional negative staining. We present a comparison of infectivity assay, reverse transcriptase assay, negative staining, and thin sectioned TEM. These assays were performed on four samples. Ultracentrifuged sediments of cleared cell-culture media were measured, fixed and processed. Thin sections were evaluated by TEM and the number of viral particles estimated by morphometric derived quantification. Retrovirus particles were easily identified and quantified when examined by TEM as compared to negative staining and correlated with the other viral assays (IF, RTA). These results demonstrate that the TEM thin section method was a superior technique to negative staining for estimating viral particle load in cell-culture supernatant. To validate further the plastic embedding with thin sectioning, we evaluated cell culture supernatants (pellets) for retroviral burden at various dilutions, from two cell lines. Morphometric determinations were made of the number of viral particles present per unit volume and compared to results obtained by infectivity assay. Since the morphometric calculation for viral density assumes even distribution of viral particles, we also evaluated and calculated viral counts on multiple thin sections taken throughout selected pellets.

Industrial experience with the detection of retroviruses.

An important aspect of the virological assessment of cell substrates used to produce biologicals is their retroviral status. In this presentation we will attempt to document some of the practical experiences of industry when testing cell substrates for retroviruses using infectivity tests, reverse transcriptase and electron microscopy. We will also explore some important aspects of the experimental design of these retrovirus assay systems and review some results from the application of these methods in model, experimental systems.

Toxicity of Echinacea purpurea. Acute, subacute and genotoxicity studies.

Single oral or intravenous doses of the expressed juice of Echinacea purpurea (EP) proved virtually non-toxic to rats and mice. After 4 weeks of oral administration in doses amounting to many times the human therapeutic dose laboratory tests and necropsy findings gave no evidence of any toxic effects in rats. Tests for mutagenicity carried out in microorganisms and mammalian cells in vitro and in mice all gave negative results. In an in vitro carcinogenicity study EP did not produce malignant transformation in hamster embryo cells.

An interlaboratory comparison of enhanced morphological transformation of Syrian hamster embryo cells cultured under conditions of reduced bicarbonate concentration and pH.

Initial studies performed in our laboratory indicated that early passage Syrian hamster embryo (SHE) cells exhibit optimal clonal proliferation when cultured in medium with a sodium bicarbonate concentration of 8.9 mM and pH of 6.70 instead of 44 mM and pH 7.35 as used previously by others. Subsequent studies indicated that morphological transformation frequency induced by benzo[a]pyrene (BP) was also enhanced at pH 6.70 compared to 7.35 and the level of enhancement was affected by cell density and duration of culture. With optimal conditions identified, the carcinogens BP, 3-methylcholanthrene, N-methyl-N'-nitro-N-nitrosoguanidine, 2-acetylaminofluorene and the non-carcinogen anthracene were tested at pH 6.70 and 7.35 in our laboratory and at Microbiological Assoc. Inc. under code. Additionally, the non-carcinogens 4-acetylaminofluorene, and caprolactam were tested in our laboratory. Results from these studies indicate that all carcinogens tested caused a significant increase in morphological transformation frequency compared to controls at pH 6.70. In contrast, only BP caused a significant increase in the morphological transformation frequency at pH 7.35. The non-carcinogens did not significantly increase the morphological transformation frequency compared to controls. Interlaboratory comparisons were in qualitative agreement despite the fact that different lots of serum and hamster cell isolates were used by the two laboratories. However, different dose-response curves for the various chemicals were observed between the two labs. It was also demonstrated that the enhanced morphological transformation frequency is not due to a decrease in culture medium osmolality or Na concentration, a condition which accompanies media with a reduced bicarbonate concentration and pH. These results demonstrate that the chemicals tested, low pH transformation of SHE cells agrees with carcinogenic potential and that assay variability is minimized. The implications of these results regarding use of the SHE cell assay as a short-term test for predicting the carcinogenic potential of chemicals are discussed.

A method for accelerating progression to a malignant phenotype in rodent cells.

This report describes a system of suspension culturing that enhances the expression of transformed cells in carcinogen-treated rodent cells and decreases the time required to observe clear evidence of the neoplastic or malignant phenotype by 2-8 wk or more. Retrovirus-infected Fischer rat embryo cells, uninfected Balb/c 3T3 mouse cells and Syrian hamster embryo cells in monolayer culture were treated with the chemical carcinogens, dimethylbenzanthracene, benzo[a]pyrene or N-methyl-N'-nitro-N-nitrosoguanidine, following a protocol appropriate to each cell type. The cultures were divided into two groups, one seeded directly onto a plastic surface, and the other suspended in liquid medium over agar before seeding onto a plastic surface. In all three cell types incluson of the suspension phase accelerated chemically induced transformation as indicated by clonogenicity in soft agarose (rat, mouse and hamster cells) and by morphological transformation and formation of tumours in athymic nude mice (hamster cells).

A selection procedure for the rapid expression of carcinogen-induced malignant transformation of retrovirus-infected Fischer rat embryo cells.

This study evaluates a procedure that accelerates the expression of the transformation of retrovirus-infected Fischer rat embryo cells. The endpoints used were anchorage-independent growth (formation of colonies in soft agarose) and formation of foci on a contact-inhibited monolayer. The cells were treated in monolayer with chemical, solvent (negative control) or medium alone for 3 days; then the chemical was removed and the cultures re-fed with medium alone for an additional 3 days. Cells in monolayer were disaggregated, suspended in liquid medium over solid agar for 4 days, disaggregated again and seeded into monolayer. After 2-4 wk without additional subculturing, cells from monolayer were seeded in soft agarose. Suspension of retrovirus-infected rat embryo cells above agar after chemical treatment resulted in rapid expression of neoplastic phenotypes. Cells treated in monolayer with the carcinogens, benzidine dichloride, dimethyl benzanthracene, 4,4-oxydianiline, 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine demonstrated colony formation in soft agarose or morphological transformation within 2-4 wk after being held in suspension for 4 days. In addition two carcinogens, benzo[a]pyrene and N-2-acetylaminofluorene and two noncarcinogens, benzo[e]pyrene and N-4-acetylaminofluorene did not induce neoplastic changes in this time period. The suspension technique may be a useful modification of this assay because it selectively amplifies neoplastic transformation after treatment with a number of chemicals.

Increased carcinogen metabolism and survival of retrovirus-infected Fischer rat embryo cells following repetitive carcinogen treatment.

The carcinogens N-2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN) often give negative results when tested in the Fischer rat embryo cell survival assay with the standard single 72-hour regimen, whereas another carcinogen, benzo(a)pyrene [B(a)P], may yield varied results between different laboratories and may require relatively high concentrations (compared with other polycyclic aromatic hydrocarbons) for a positive result to occur. Enhanced survivals (compared with controls) were 56% or less with these carcinogens. In place of the standard single 72-hour treatment with test chemical, the cells were exposed to three consecutive 24-hour treatments. The amount of B(a)P metabolized during the last of the three 24-hour treatment periods was 3.2 times greater than that during the first 24-hour period, indicating that an induction effect occurred. Furthermore, the total amount of metabolites of B(a)P formed with repetitive treatments was 2.1 times greater than with a single 72-hour treatment. The total amount of AAF metabolites formed with repeated treatments was 1.6 times greater than with the single treatment regimen, although no induction effect was observed between treatment periods. Survival enhancement with the repetitive regimen increased to 181% with B(a)P, 172% with AAF, and 188% with DEN. With benzo(e)pyrene, anthracene, and pyrene, enhanced survival was 14% or less following the single treatment regimen and did not increase following repetitive treatments. When the carcinogen cinnamyl anthranilate was tested using repetitive treatments, survival enhancement was more than 100% at three of six doses, versus less than 0% when the standard single treatment regimen was used.

Chemical enhancement of survival in aggregation of retrovirus-infected rat cells: an interlaboratory comparison.

This study provides a comparative evaluation among three independent laboratories of the responses to 16 chemicals in the retrovirus (Rauscher leukemia) infected Fischer rat embryo (RIFRE) cell-survival-in-aggregation assay. When suspended in liquid media above an agar base, control cells showed a rapid decline in cell survival, whereas cells that had previously been treated with chemical carcinogen survive in suspension longer than control cells. The endpoint, survival in aggregation, is measured by counting viable cells dissociated from aggregates in suspension for 4 days. By modifying previously reported procedures, we have improved the system so that a clear differential (positive or negative) response is achieved by cells treated with either a known carcinogen or known noncarcinogen. Using procedures designed to minimize assay variability, replicate assays were performed and the data analyzed for inter- and intralaboratory concordance. The RIFRE cell-survival-in-aggregation assay demonstrated a high degree of interlaboratory reproducibility in assessing the overall positive or negative responses of known carcinogens and noncarcinogens, and good qualitative reproducibility in assessing compounds tested under code. The assay could discriminate between known carcinogens and noncarcinogens. All chemicals were tested without the addition of a metabolic activation system. Cells exhibiting carcinogen-induced enhancement of survival in aggregation, when plated back onto a solid substrate and carried in culture, subsequently expressed transformation-associated changes in their cellular morphology, growth in semisolid media, and tumorigenicity in nude mice. These results indicate that retrovirus-infected Fischer rat embryo cells detect a carcinogen-mediated early event that progresses to neoplastic phenotypes. Survival in aggregation appears to require the presence of the exogenous retrovirus, since uninfected cells did not show a differential survival response when carcinogen-treated, noncarcinogen-treated, or control cells were compared. This system provides a reproducible method of detecting carcinogenic chemicals based on their ability to induce enhanced survival in aggregation of treated cells.

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

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 metabolize N-2-acetylaminofluorene to 2-aminofluorene and N-hydroxy-acetylaminofluorene, products that transform hamster embryo cells. Large amounts of N-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 of N-hydroxy-acetylaminofluorene were formed after incubation of N-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 with N-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.

Use of the hamster embryo cell transformation assay to detect metabolic activation of N-2-acetylaminofluorene by intact organ cells.

N-2-Acetylaminofluorene (AAF), a potent carcinogen in a variety of animal species and organs, was used to determine the metabolic capabilities of isolated organ cells in transformation as well as biochemical studies. Cells isolated from liver, lung, small intestine, kidney and bladder were compared with hamster embryo fibroblasts (target cells in the transformation studies) and rat mammary fibroblasts in all studies. In addition to studying AAF activation by the cells, we also determined the levels of whole-cell binding. Liver, kidney, small intestine and lung cells from hamsters, and liver, kidney and lung cells from rats showed high levels of AAF metabolism to 2-aminofluorene and N-hydroxy-2-acetylaminofluorene. The highest levels of covalent binding to intact cells were seen with the same cell types. These cells were also effective in activating AAF to a form which transformed hamster embryo cells. Cells isolated from a variety of organs can activate AAF as evidenced by the metabolites which are formed and by the levels of whole cell binding. Furthermore, hamster embryo cells are transformed when co-incubated with a variety of organ cells and AAF.

Metabolic activation, detoxification and transformation studies with benzo[a]pyrene using rat and hamster organ cells in vitro.

The mechanism of benzo[a]pyrene (BP) by cultured kidney, liver, small intestine, lung and trachea from male Syrian golden hamsters and Sprague-Dawley rats was compared. The metabolic capacity of intact cell systems was assessed by determining the formation of organic-solvent and water-soluble metabolites, levels of covalent binding and levels of BP metabolites following separation using high-pressure liquid chromatography. Hepatocytes from both species metabolized three to four times more BP than the other cells studied. Formation of water-soluble metabolites by hepatocytes was at least 10 times that of the other cell types. Generally, hamster cells had greater metabolic capability than rat cells. Levels of covalent binding of metabolites of BP were at least 10 times greater in hepatocytes (uninduced) from both species than in the other cell types. Binding to hepatocytes and hamster embryo cells increased with incubation time coinciding with an increase in the formation of water-soluble and diol metabolites. High levels of water-soluble metabolites accompanied high levels of covalent, whole-cell binding. Hamster embryo cells are transformed by BP without an exogenous metabolic activation system. The presence of hamster and rat hepatocytes inhibited transformation of hamster embryo cells by BP. This inhibition of transformation correlated with the increased rate of formation of water-soluble detoxification metabolites by hepatocytes from both species. The high rate of formation of water-soluble products by intact hepatocytes reflects the in vivo activity of liver cells. Use of hepatocytes in short-term tests allows a system for carcinogen metabolism more closely representing that which is present in the whole animal.

Greater effectiveness of hepatocyte and liver S9 preparations from hamsters than rat preparations in activating N-nitroso compounds to metabolites mutagenic to Salmonella.

A comparison was made of the ability of liver S9 and hepatocyte preparations from noninbred Syrian golden hamsters and noninbred Sprague-Dawley rats to metabolically activate a number of nitroso compounds in the Salmonella mutagenesis assay. The liver S9 and hepatocyte preparations from hamsters were consistently more effective than were preparations from rats in metabolizing nitrosodimethylamine (NDM), nitrosodiethylamine, nitrosodiallylamine, nitrosopyrrolidine (NP), nitrosomorpholine (NM), nitrosodiethylmethylurea (NDEMU), and nitrosodimethyl-ethylurea (NDMEU) to mutagenic forms. The use of hamster S9 preparations with NP and NM resulted in up to 14 times the number of revertant colonies obtained with rat preparations; in the presence of hamster hepatocytes, up to 32 times the number of revertants were obtained. The S9 preparations from male hamsters not treated with the enzyme inducers phenobarbital and Aroclor 1254 were more effective than were those from female hamsters for activating NP, NM, and NDM, NDEMU and NDMEU, which have been reported to be carcinogens but not mutagens, were mutagenic in the presence of induced liver S9 or hepatocyte preparations from hamsters but not from rats. When tested with any of the S9 or hepatocyte preparations, nitrosodiphenylamine and nitrosomethylaniline, also reported to be carcinogens but not mutagens, gave no mutagenic responses. Nitrosodioctyl-amine, which has been reported to be noncarcinogenic, was also not mutagenic.

Assessment of environmental contamination associated with a mammalian cell transformation assay.

To estimate worker exposures to, and environmental contamination from, test chemicals and organic solvents used in an in vitro assay to assess the carcinogenic potential of chemicals, sodium fluorescein, a noncarcinogenic fluorescent material, was dissolved in tissue culture medium used to maintain early passage hamster embryo cells. Personal an environmental samples were taken over a 14-d period. The assay was performed according to standard procedures in a ventilated glove box or laminar flow safety cabinet. Considerably more than 99% of the chemical contamination found was recovered from the interiors of the glove box and hood and from disposable equipment. Contamination outside the containment units (less than 1 microgram) resulted from intralaboratory transport of chemicals, treated cultures, and contaminated equipment. We conclude that the standard operating particles and procedures provided adequate safeguards for personnel and the environment.

Metabolic activation by hamster and rat hepatocytes in the Salmonella mutagenicity assay.

Intact and homogenized hepatocytes from untreated or Aroclor 1254-treated male and female noninbred Sprague-Dawley rats and noninbred Syrian golden hamsters were compared for their ability to metabolize chemicals in the Salmonella-mammalian microsome mutagenesis assay. The following chemicals were used: two aromatic amines, 2-amino-anthracene and N-2-fluorenylacetamide; two polycyclic aromatic hydrocarbons, 3-methylcholanthrene and benzo[a]pyrene (BP); and one nitrosamine, diethylnitrosamine (DENA). With one exception, hepatocytes from hamsters were more active than were hepatocytes from rats in the activation of these mutagens. The homogenized preparations from Aroclor 1254-treated rats were slightly more active with BP than was the equivalent hamster preparation. Intact hepatocytes from Aroclor 1254-treated hamsters were more efficient at metabolizing the aromatic amines and DENA, whereas homogenates were more effective with the hydrocarbons. Results were similar with the rat preparations, except that only large quantities of Aroclor 1254-treated intact male rat hepatocytes appeared to activate DENA. These results suggest that, in the choice of an activation system, the kind of chemical being evaluated should be considered.

Correlation between transformation potential and inducible enzyme levels of hamster embryo cells.

When benzo(a)pyrene was used to evaluate the transformability of 129 hamster embryo cell preparations from pooled or individual embryos, approximately 50% of the cultures were transformable. A transformable and a non-transformable cell culture were further tested with other carcinogens (3-methylcholanthrene [MCA], benzyl chloride, ethyl-p-toluenesulfonate, 2-naphthylamine, and aflatoxin B1). The transformable culture responded to all of the carcinogens while the non-transformable culture always gave negative results. Aryl hydrocarbon hydroxylase (AHH) and epoxide hydrase (EH) levels were compared in the two cell cultures using beta-naphthoflavone (BNF), benz(a)anthracene (BA), sodium phenobarbital (PB) or MCA as microsomal enzyme-inducing agents. It was found that AHH levels and the degree of induction following treatment of the cells with BNF or BA were consistently higher in the transformable than in the non-transformable cells following treatment with either BNF, BA, PB or MCA. Inducible AHH and EH levels might, therefore, be useful as predictors of the transformation potential of hamster embryo cell cultures.

Effect of fetal bovine serum on 3-methylcholanthrene-induced transformation of hamster cells in vitro.

Eighteen lots of fetal bovine serum were tested for their ability to support clonal growth and 3-methylcholanthrene-induced morphological transformation of hamster embryo cells in vitro. Most of them supported cloning efficiencies of over 11%. However, cloning efficiency alone was an inadequate criterion for selecting serum for transformation studies, since no transformation was observed with some lots, even though their cloning efficiencies were over 16%. This shows the importance of pretesting serum for its ability to support morphological transformation before it is used in mammalian cell carcinogenesis tests.