Morphological transformation of C3H/M2 mouse fibroblasts by, and genotoxicity of, extracts of human milk

Breast cancer may be initiated by environmental/dietary agents and human milk may act as an ex vivo indicator of in vivo exposure of mammary epithelial cells to genotoxins. Extracts of human milk from UK-resident women (n=7) were tested for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Genotoxicities were assessed in the Salmonella typhimurium reverse-mutation assay in the presence of S9 using strains TA1538 and YG1019, and in metabolically-competent human MCL-5 cells with the micronucleus and with the alkaline single cell gel electrophoresis (comet) assays. Two of the seven extracts were inactive in the transformation assay both in the presence or absence of S9, two appeared to be equally transforming either in the presence or absence of S9, and two other extracts induced increased transformation frequencies in the presence of S9. A seventh extract, tested only in the absence of S9, was inactive. Extracts were either active or inactive in at least three of the four tests applied. Four extracts were active or inactive in all four tests. The results suggest that human milk could be used as a resource for investigations of the as-yet-unidentified transforming agents previously detected in mammary lipid.     

An endogenous protein inhibitor of DNA polymerase alpha in normal and neoplastic rat mammary tissues

Extracts of whole tissue or isolated nuclei from lactating rat mammary gland that has diminished cell replication capacity were more active than the corresponding extracts of pregnant rat mammary gland that contains actively replicating cells in causing a dose-dependent inhibition of DNA polymerase alpha in vitro. Purification of the inhibitor from both tissue and nuclear extracts using a sequence of Sephacryl S200, DEAE-cellulose and CM52 columns confirmed the above assay results. Using the same assay and purification procedures, both tissue and nuclear extracts from the rapidly growing transplanted R3220AC mammary tumors exhibited very little or no inhibitor activity. The partially purified mammary inhibitor (mol. wt of 155kD, high A280 nm/A260 nm ratio, heat labile) was equally inhibitory to the purified DNA polymerase alpha from either R3230AC tumor or calf thymus, and to the nuclear matrix bound DNA polymerase alpha of R3230AC tumor.     

The presence of genotoxic and bioactive components in indigo dyed fabrics--a possible health risk?

Extracts of pure cotton and jeans fabrics were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100. The vat dye indigo, technical grade as well as 98% and greater than 99.5% pure, was also tested for mutagenicity. Synthetic indigo, indirubin and isatin were tested for TCDD receptor affinity in competition experiments in vitro. The mutagenicity of the extracts was associated with the cotton denim and nondyed cotton gave only marginal effects. The mutagenicity of the indigo dyed fabrics was dependent on type and treatment of the fabrics. Extracts of both bleached and nonbleached jeans gave mutagenic effects on TA98 +/- S9 and TA100 +/- S9. The greatest effects were seen in the presence of S9. Bleaching gave an additional increase in the mutagenicity in the absence of S9. Normal washing of the fabrics after bleaching reduced the mutagenicity. Synthetic indigo of technical grade or 98% pure showed mutagenic effects, especially on TA98 + S9. Further purification to 99.5% reduced the mutagenicity to 24 revertants/mg (6.2 rev/mu mole). Considering the amount of indigo in the extracts and its low mutagenicity, the genotoxicity of jeans extracts must be caused by other unknown components. However, indigo showed a high (Kd = 1.9 nM) affinity for the Ah or TCDD receptor. Indigo can therefore still be a potential health risk either by eliciting toxic effects of other compounds or by being a nongenotoxic carcinogen. The worldwide use of jeans with a possible exposure of a large population to genotoxic and biologically active components emphasizes the need for a more thorough characterization of these effects.     

Release of mutagens from finished leather

Extracts of a leather widely used in the furniture and dress-making industries were tested for their mutagenic activity in the Salmonella/microsome assay. Extracts obtained after vigorous treatment of leather samples in a Soxhlet apparatus with toluene or ethanol were mutagenic in strain TA98 of S. typhimurium in the absence of S9 mix. The analysis of extracts of leather at various intermediate stages of processing showed that the mutagenic activity appeared after the coloration process. The responsible compound was identified to be an azo dye (Color Index: Acid Brown 83) whose mutagenic potency was about 4 revertants/micrograms.     

In vitro genotoxicity of para-phenylenediamine and its N-monoacetyl or N,N'-diacetyl metabolites

para-Phenylenediamine (PPD), a widely used ingredient of oxidative hair dyes, is converted by human hepatocytes and in the human epidermis, or after topical application to rats, to its N-monoacetylated (MAPPD) and/or N,N'-diacetylated (DAPPD) derivatives. We investigated in vitro genotoxic properties of PPD, MAPPD and DAPPD in the Ames test, the micronucleus test (MNT) in human lymphocytes and the mouse lymphoma assay (Hprt locus, PPD only). Given that MAPPD and DAPPD are actual human skin and hepatic metabolites of PPD and represent the substances to which humans are systemically exposed, they were tested in the absence of metabolic activation. In the Ames test, PPD was slightly mutagenic in Salmonella typhimurium strain TA98 in the presence of a rat liver metabolic activation system (S-9), whereas MAPPD and DAPPD were negative in all strains. When tested up to toxic doses, PPD did not induce mutation at the Hprt locus of L5178Y mouse lymphoma cells in two independent experiments, either in the absence or presence of S-9, suggesting that PPD is non-mutagenic in mammalian cells. In the in vitro micronucleus test, PPD induced micronuclei (MN) in cultured human peripheral blood lymphocytes (HL) in the presence of S-9, when tested following 24-h PHA stimulation. No increases in MN frequency were observed in the absence of S-9, when tested following 24-h PHA stimulation. However, PPD induced MN both in the absence and presence of metabolic activation, when tested following 48-h PHA stimulation. In contrast, MAPPD and DAPPD did not induce MN in HL when tested up to 10mM concentrations or to their limit of solubility, respectively, after either 24- or 48-h stimulation. In conclusion, the results of the Ames and MN tests confirm that PPD has a slight genotoxic potential in vitro, although it was non-mutagenic in mammalian cells. Given that MAPPD and DAPPD were negative in the Ames and the MN tests, these acetylated conversion products are considered to be detoxified metabolites that are biologically less reactive than the parent molecule PPD.     

In vitro mutagenicity and cell-transformation screening of N-(2,3-epoxy-propyl)-phthalimide.

N-(2,3-Epoxy-propyl)-phthalimide (EPP) was tested for genetic activity in the Salmonella/microsome mutagenicity test. Concentration-dependent mutagenicity was demonstrated in S. typhimurium strains TA1535, TA1537 and TA100 with and without rat S9. It was inactive in strain TA1538, and active without rat S9 in TA98 at the high dose. EPP induced 6-thioguanine-resistant mutants of Chinese hamster ovary cells in the absence of an exogenous activating system. EPP produced dose-dependent enhancement of SA7 virus transformation of primary hamster-embryo cells, and transformed secondary hamster-embryo cells in a non-dose-related fashion. At a dose of 5 g/kg p.o. or i.m., EPP was inactive in the host-mediated assay using C57Bl/6XC3H mice and S. typhimurium strain TA1535. Murine testicular DNA synthesis was not inhibited by oral administration of EPP at 1000 mg/kg.     

Antioxidant and antigenotoxic activities of Angelica keiskei, Oenanthe javanica and Brassica oleracea in the Salmonella mutagenicity assay and in HCT116 human colon cancer cells

Epidemiological studies indicate that consumption of green-yellow vegetables rich in chlorophyll, vitamin C, vitamin E, and carotenoids reduce the risk of cancer. We sought to examine the antigenotoxic and antioxidant properties of chlorophyll-rich methanol extracts of Angelica keiskei, Oenanthe javanica, and Brassica oleracea (kale). In the Salmonella mutagenicity assay, A. keiskei caused dose-dependent inhibition against three heterocyclic amine mutagens in the presence of S9, O. javanica was antimutagenic only at the highest concentration in the assay (2 mg/plate), and B. oleracea showed no consistent inhibitory activity at non-toxic levels. None of the extracts were effective against three direct-acting mutagens in the absence of S9. Extracts of A. keiskei and, to a lesser extent O. javanica, inhibited two of the major enzymes that play a role in the metabolic activation of heterocyclic amines, based on ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase assays in vitro. All three plant extracts were highly effective in assays which measured ferric reducing/antioxidant power, oxygen radical absorbance capacity, and Fe2+/H2O2-mediated DNA nicking. Finally, using the 'comet' assay, all three plant extracts protected against H2O2-induced genotoxic damage in human HCT116 colon cancer cells. These findings provide support for the antigenotoxic and antioxidant properties of chlorophyll-rich extracts of A. keiskei, O. javanica, and B. oleracea, through mechanisms that include inhibition of carcinogen activation and scavenging of reactive oxygen species.     

beta-carotene as enhancer of cell transforming activity of powerful carcinogens and cigarette-smoke condensate on BALB/c 3T3 cells in vitro

We report the ability of beta-carotene (betaC) to affect the cell transforming activity of 3-methylcholanthrene (3-MCA), benzo(a)pyrene (B(a)P) and cigarette-smoke condensate (TAR) in an in vitro medium-term (approximately 8 weeks) experimental model utilizing BALB/c 3T3 cells. Different experimental schedules were performed either in the presence or absence of betaC: (i) cultures treated for 72 h with each chemical (acute treatment), (ii) cultures grown in presence of each chemical for the whole period of the experiment (chronic treatment). These procedures suggested a possible cocarcinogenic potential of the carotenoid following interactions with other chemicals mimicking continuous human exposition to several xenobiotics. Although the pigment did not show any cell transforming potential when tested alone either in acute or chronic treatment, it did augment that of other tested agents. Induction of cell transformation by B(a)P was markedly enhanced by the presence of this carotenoid in either acute or chronic treatment. Only in presence of betaC, was TAR able to significantly act as a cell transforming agent in prolonged, chronic treatment of cultures. Enhanced cell transformation activity could be due to the boosting effect of betaC on P450 apparatus. Indeed, elsewhere we have found that the latter increased the ratio of formation of diol epoxide carcinogenic metabolites of B(a)P as well as other carcinogens present in TAR. By contrast, no differences of cell transforming activity of 3-MCA, an ultimate carcinogen, were seen either in the presence or absence of betaC under the various experimental conditions. These data, which are in keeping with the cocarcinogenic potential of betaC, may help to explain the unexpected lung cancer increases obtained in chemoprevention trials in heavy smokers supplemented with the isoprenoid. Our findings also highlight the potential risk to humans derived from interactions among xenobiotics present in the environment.     

Keratinocyte growth-promoting activity from human placenta

Extracts of term human placenta were tested for enhancement of proliferative growth of primary cultures of human keratinocytes. Saline extracts or supernatants from homogenates were dialyzed extensively, lyophilized, and tested in subcultures of keratinocytes in MCDB 153 medium with 0.1 mM Ca++ containing only defined supplements (insulin, hydrocortisone, transferrin, ethanolamine, phosphoethanolamine). Cells plated in the absence of EGF at moderately high densities (1000-3000 cells per cm2) formed colonies and grew in the presence of placental extract at 25-500 micrograms/ml. Extracts of cord serum or maternal serum were inactive, suggesting that the activity is derived from placental tissue. The activity is not EGF, since the activity in the placental extract, unlike EGF, did not promote growth at low cell density, was synergistic with EGF under some conditions, and did not produce changes in colonial morphology which occurred in the presence of EGF. Unlike keratinocyte growth-promoting activity in bovine hypothalamic extract, the activity is non-dialyzable and is destroyed at 100 degrees C. Placental extract could not replace any of the defined components of the medium and is therefore distinct from them. The presence of activity in the placenta with distinctive properties suggests that this is a previously undescribed material with growth-promoting properties for epithelium.     

Mutagenicity of extracts from typewriter ribbons and related items

Extracts of typewriter ribbons and carbon papers were found to be mutagenic in the Salmonella/microsome assay with strain TA98. Fractionation of ribbon extracts indicates that at least 2-3 different classes of mutagenic component are present in these extracts. Nitro-containing compounds may be responsible for the high mutagenicity observed for some of the ribbon extracts in the absence of S9. The results indicate that impurities in the products may be causing part of the mutagenic effect.     

An evaluation of three pesticides: Piritione,supercypermethrin and metolachlor in transformation bioassays of BHK21 and hamster embryo cells

In a study of potential carcinogenicity of pesticides, Piritione, metolachlor (in the form of Dual and VUCHT 524) and Supercypermethrin (in the form of Supercypermethrin EC and Supercypermethrin TP) were assayed for induction of anchorage independent growth of BHK21 cells and morphological transformation of Syrian hamster embryo cells. The activity of these substances in both transformation assays was compared to the activity of the direct-acting ultimate carcinogen N-methyl-N-nitrosourea. In comparison to the very strong transforming activity of N-methyl-N-nitrosourea all pesticides tested with or without S9 fraction manifested a very weak, weak, medium or strong effect. The ability to induce anchorage independent growth was graded as follows: Dual < Supercypermethrin EC < Supercypermethrin TP Piritione < VUCHT 524. Results of Syrian hamster embryo cell transformation assay were very similar to the BKH21 transformation assay. VUCHT 524 strongly induced transformation whereas Dual was inactive. Piritione and Supercypermethrin EC and Supercypermethrin TP elicited a slight but significant positive response.     

Growth-promoting actions of extracts from mouse submaxillary glands on human endothelial cells in culture.

Extracts of submaxillary glands from two different strains of inbred mice were mitogenic for human endothelial cells in culture. The mitogenic activity of extracts from glands of males of the SWR/J and C57BL/10J strains were equivalent, and the growth stimulating effect was unrelated to renin or esteroproteolytic activity. Mitogenic activity in extracts from SWR/J females was less than that from males, and extracts from C57BL/10J females were inactive. The polypeptide growth factors, epidermal (EGF) and fibroblast (FGF) growth factors, also stimulated replication of endothelial cells. Cells from either umbilical arteries or veins responded to submaxillary extracts, EGF, or FGF with a similar increase in cell number, increase in protein and enhanced uptake of 3H-thymidine. The proliferative response was associated with decreased activity of angiotensin I converting enzyme which is localized on the endothelial surface. Nerve growth factor (NGF) was not mitogenic for endothelial cells. Extracts of submaxillary glands from male mice of either strain contained approximately 20 times more EGF than extracts from females, as determined by immunodiffusion. Mitogenic activity of the extracts was completely inhibited by antiserum to EGF, suggesting that the active component of these preparations is EGF.     

Balbc/3T3 cell transformation response to extracts of organic air samples as seen by their survival in aggregate form

Extracts of organic matter from samples of airborne particulate matter have been shown to possess components capable of transforming mammalian cells. This study was done to determine if Balbc/3T3 cells exposed to extracts of air samples could, unlike their normal counterparts, in the absence of a surface for attachment, divide on agar to form aggregates, and if these cells would demonstrate a dose-response phenomenon. Untreated and solvent treated control cells failed to form large aggregates and showed a decline in viable cell number over a 6-day period. Cells treated with either cyclohexane or acetone extracts of airborne particulate matter showed a dose-response increase in cell number along with the formation of progressively larger aggregates, findings similar to those seen with the positive, benzo[a]pyrene (BaP), control. Furthermore, these findings are in direct agreement with those in the simultaneously performed standard cell transformation assay which required 21 days to perform. Results show that survival in aggregate form is a rapid in vitro test system capable of detecting potentially carcinogenic activity in complex environmental mixtures.     

Evaluation of chemopreventive agents for genotoxic activity

We conducted genetic toxicity evaluations of 11 candidate chemopreventive agents with the potential for inhibiting carcinogenesis in humans at increased risk of cancer. The compounds were evaluated for bacterial mutagenesis in the Salmonella-E. coli assay, for mammalian mutagenesis in mouse lymphoma cells, for chromosome aberrations in Chinese Hamster Ovary (CHO) cells, and for micronucleus induction in mouse bone marrow. Tested agents were indole 3-carbinol (I3C), bowman-birk inhibitor concentrate (BBIC), black tea polyphenols (BTP), farnesol, geraniol, l-Se-methylselenocysteine (SeMC), 5,6-dihydro-4H-cyclopenta[1,2]-dithiol-3-thione(DC-D3T), 4'-bromoflavone, 2,5,7,8-tetramethyl-(2R-[4R,8R,12-trimethyltridecyl] chroman-6-yloxy) acetic acid (alpha-TEA), SR13668 (2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo[2,3-b] carbazole and SR16157 (3-O-sulfamoyloxy-7alpha-methyl-21-(2-N,N-diethylaminoethoxy)-19-norpregna-1,3,5(10)-triene). All these agents, except I3C and BTP, were negative in the Salmonella-E. coli assay in the presence and absence of metabolic activation (S9). I3C and BTP induced a weak mutagenic response in the presence and absence of S9 with strains TA100 and TA98, respectively. Of the three compounds tested in the mouse lymphoma assay (I3C, BBIC, and BTP), only BTP was mutagenic in the presence of S9. In the chromosomal aberration assay, of the 8 compounds that were tested, 4'-bromoflavone elicited a positive response in the absence of S9 only, while SR16157 was positive in the presence of S9. The results with geraniol remain inconclusive. I3C, BBIC and BTP were not tested in the chromosomal aberration assay. None of the 11 agents induced micronuclei in mouse bone marrow erythrocytes.     

Genotoxicity of the cancer chemopreventive drug candidates CP-31398, SHetA2, and phospho-ibuprofen

The genotoxic activities of three cancer chemopreventive drug candidates, CP-31398 (a cell permeable styrylquinazoline p53 modulator), SHetA2 (a flexible heteroarotinoid), and phospho-ibuprofen (PI, a derivative of ibuprofen) were tested. None of the compounds were mutagenic in the Salmonella/Escherichia coli/microsome plate incorporation test. CP-31398 and SHetA2 did not induce chromosomal aberrations (CA) in Chinese hamster ovary (CHO) cells, either in the presence or absence of rat hepatic S9 (S9). PI induced CA in CHO cells, but only in the presence of S9. PI, its parent compound ibuprofen, and its moiety diethoxyphosphoryloxybutyl alcohol (DEPBA) were tested for CA and micronuclei (MN) in CHO cells in the presence of S9. PI induced CA as well as MN, both kinetochore-positive (Kin+) and -negative (Kin-), in the presence of S9 at ≤100μg/ml. Ibuprofen was negative for CA, positive for MN with Kin+ at 250μg/ml, and positive for MN with Kin- at 125 and 250μg/ml. DEPBA induced neither CA nor MN at ≤5000μg/ml. The induction of chromosomal damage in PI-treated CHO cells in the presence of S9 may be due to its metabolites. None of the compounds were genotoxic, in the presence or absence of S9, in the GADD45α-GFP Human GreenScreen assay and none induced MN in mouse bone marrow erythrocytes.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 microg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 microg/ml for 3 and 20 h in the absence of S9, and for 3h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 microg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 microg/kg, the last dose being administered 2h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Novel multialveolar epithelial structures from rabbit mammary gland that synthesize milk specific fatty acids in response to prolactin

Multialveolar mammary epithelial structures have been prepared from rabbit mammary gland by treating the tissue with collagenase plus hyaluronidase. These structures synthesize milk specific fatty acids when cultured with physiological concentrations (0.5 micrograms/ml) of prolactin in the presence of insulin and corticosterone. They have many of the advantages but few of the disadvantages of either mammary explants or primary cells in culture. For example, they are easily prepared in large numbers and respond to prolactin in culture even in the absence of serum or other tissue extracts. Because their level of organization is intermediate between that of explants and single cells, they provide a complementary system for studies on mammary differentiation.     

Genotoxicity of soil from farmland irrigated with wastewater using three plant bioassays.

Three well known plant bioassays, the Allium root chromosome aberration (AL-RAA) assay, the Tradescantia micronucleus (Trad-MCN) assay, and the Tradescantia stamen hair (Trad-SHM) mutation assay were validated in 1991 by the International Programme on Chemical Safety (IPCS) under the auspices of the World Health Organization, and the United Nations Environment Programme (UNEP). These plant bioassays have proven to be efficient tests for chemical screening and especially for in situ monitoring for genotoxicity of environmental pollutants. As a result of this validation study, standard protocols of these three plant bioassays were used by some of the 11 participating countries in the IPCS to carry on genotoxicity tests on air, water and soil as a follow up activity. In the city of Queretaro, Mexico, wastewater coming from both industrial and domestic sources and without any treatment is used to irrigate the farm crops, polluting the soil. Potentially the pollutants could reach the food chain. For the above reason, soil irrigated with wastewater was sampled and monitored for the presence of genotoxic agents using the above three bioassays. Extracts from soil samples were made using distilled water and organic solvents by shaking the sample for about 12 h under a relatively low temperature (15-20 degrees C). Plant cuttings of Tradescantia or the roots of Allium were treated by submerging them in the extracts. Three replicates of each sample were analyzed in each of the three bioassays. Extracts using DMSO, ethanol and distilled water tested positive in the three bioassays and there were no differences for the genotoxicity of the extracts with the different solvents.     

Transgene expression in mammary glands of newborn rats

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals. © 1996 Wiley-Liss, Inc.