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Carbon nanotubes (CNTs) belong to a specific class of nanomaterials with unique properties. Because of their anticipated use in a wide range of industrial applications, their toxicity is of increasing concern. In order to determine whether specific physicochemical characteristics of CNTs are responsible for their toxicological effects, we investigated the cytotoxic and genotoxic effects of eight CNTs representative of each of the commonly encountered classes: single-SW-, double- DW-, and multiwalled (MW) CNTs, purified and raw. In addition, because most previous studies of CNT toxicity were conducted on immortalized cell lines, we decided to compare results obtained from V79 cells, an established cell line, with results from SHE (Syrian hamster embryo) cells, an easy-to-handle normal cell model. After 24 hours of treatment, MWCNTs were generally found to be more cytotoxic than SW- or DWCNTs. MWCNTs also provoked more genotoxic effects. No correlation could be found between CNT genotoxicity and metal impurities, length, surface area, or induction of cellular oxidative stress, but genotoxicity was seen to increase with CNT width. The toxicity observed for some CNTs leads us to suggest that they might also act by interfering with the cell cycle, but no significant differences were observed between normal and immortalized cells. [ABSTRACT FROM AUTHOR]

Study Objectives: In regard to nuclear medicine literature reporting lung uptake of colloidal radiopharmaceuticals in patients with liver diseases, it has been hypothesized that liver abnormalities could trigger induction of pulmonary intravascular macrophages (PIMs) in humans normally lacking them. Recently, experimental induction of PIMs in rats in which they are not normally prevalent has been demonstrated to be at the origin of pulmonary hemodynamic alterations with an increased susceptibility to ARDS. If such induction may occur in humans, the risk of pulmonary hemodynamic alterations has to be considered and detected. This study demonstrates in a rodent model of biliary cirrhosis that scintigraphy of phagocytic function as commonly used for liver exploration is a suitable strategy for staging PIM development.Design: Sixty rats were randomized as follows: bile duct section (n = 40), sham operation (n = 10), and no operation (n = 10). The rats were submitted to scintigraphy of phagocytic function every 5 days over 35 days for the assessment of radiocolloid uptake within lung and liver. At day 35, radioactivity of blood was counted and immunohistochemistry was performed on lung specimens.Results: As disease progressed, radiopharmaceutical uptake decreased within the liver, while increasing considerably in the lung. At day 35, lung uptake averaged about 66% as compared to 3% before surgery. Lung histologic findings revealed numerous intravascular mononuclear cells closely related to the monocyte-macrophage lineage.Conclusion: Scintigraphy of phagocytic function commonly used for liver scanning could be a suitable strategy for the diagnosis of the induction of PIMs under pathologic situations. [ABSTRACT FROM AUTHOR]

To date, long-term rodent carcinogenesis assays are the only assays recognized by regulators to assess non-genotoxic carcinogens, but their reliability has been questioned. In vitro cell transformation assays (CTAs) could represent an interesting alternative to animal models as it has the advantage of detecting both genotoxic and non-genotoxic transforming chemicals. Among them, Bhas 42 CTA uses a cell line that has been transfected with the oncogenic sequence v-Ha-ras. This sequence confers an "initiated" status to these cells and makes them particularly sensitive to non-genotoxic agents. In a previous work, transcriptomic analysis revealed that the treatment of Bhas 42 cells with transforming silica (nano)particles and 12-O-tetradecanoylphorbol-13-acetate (TPA) commonly modified the expression of 12 genes involved in cell proliferation and adhesion. In the present study, we assess whether this signature would be the same for four other soluble transforming agents, i.e. mezerein, methylarsonic acid, cholic acid and quercetin. The treatment of Bhas 42 cells for 48 h with mezerein modified the expression of the 12 genes of the signature according to the same profile as that of the TPA. However, methylarsonic acid and cholic acid gave an incomplete signature with changes in the expression of only 7 and 5 genes, respectively. Finally, quercetin treatment induced no change in the expression of all genes but exhibited higher cytotoxicty. These results suggest that among the transforming agents tested, some may share similar mechanisms of action leading to cell transformation while others may activate different additional pathways involved in such cellular process. More transforming and non-transforming agents and gene markers should be tested in order to try to identify a relevant gene signature to predict the transforming potential of non-genotoxic agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Exposure of consumers to aluminum-containing nanomaterials (Al NMs) is an area of concern for public health agencies. As the available data on the genotoxicity of Al 2 O 3 and Al 0 NMs are inconclusive or rare, the present study investigated their in vitro genotoxic potential in intestinal and liver cell models, and compared with the ionic form AlCl 3 . Intestinal Caco-2 and hepatic HepaRG cells were exposed to Al 0 and Al 2 O 3 NMs (0.03 to 80 μg/cm 2 ). Cytotoxicity, oxidative stress and apoptosis were measured using High Content Analysis. Genotoxicity was investigated through γH2AX labelling, the alkaline comet and micronucleus assays. Moreover, oxidative DNA damage and carcinogenic properties were assessed using the Fpg-modified comet assay and the cell transforming assay in Bhas 42 cells respectively. The three forms of Al did not induce chromosomal damage. However, although no production of oxidative stress was detected, Al 2 O 3 NMs induced oxidative DNA damage in Caco-2 cells but not likely related to ion release in the cell media. Considerable DNA damage was observed with Al 0 NMs in both cell lines in the comet assay, likely due to interference with these NMs. No genotoxic effects were observed with AlCl 3 . None of the Al compounds induced cytotoxicity, apoptosis, γH2AX or cell transformation., (Copyright © 2021. Published by Elsevier Ltd.)

Synthetic amorphous silica nanoparticles (SAS) are used widely in industrial applications. These nanoparticles are not classified for their carcinogenicity in humans. However, some data still demonstrate a potential carcinogenic risk of these compounds in humans. The Bhas 42 cell line was developed to screen chemicals, as tumor-initiators or -promoters according to their ability to trigger cell-to-cell transformation, in a cell transformation assay. In the present study, we performed unsupervised transcriptomic analysis after exposure of Bhas 42 cells to NM-203 SAS as well as to positive (Min-U-Sil 5® crystalline silica microparticles, and 12-O-tetradecanoylphorbol-13-acetate) and negative (diatomaceous earth) control compounds. We identified a common gene signature for 21 genes involved in the early stage of the SAS- Min-U-Sil 5®- or TPA-induced cell transformation. These genes were related to cell proliferation (over expression) and cell adhesion (under expression). Among them, 12 were selected on the basis of their potential impact on cell transformation. RT-qPCR and western blotting were used to confirm the transcriptomic data. Moreover, similar gene alterations were found when Bhas 42 cells were treated with two other transforming SAS. In conclusion, the results obtained in the current study highlight a 12-gene signature that could be considered as a potential early "bio-marker" of cell transformation induced by SAS and perhaps other chemicals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Exposure to aerosols from metalworking fluids (MWF) has previously been related to a series of adverse health outcomes (eg, cancer, respiratory diseases). Our present epidemiological study focuses on occupational exposures to MWF and a panel of exposure and effect biomarkers. We hypothesize that these health outcomes are caused by particle exposure that generates oxidative stress, leading to airway inflammation and ultimately to chronic respiratory diseases. We aimed to assess whether MWF exposure, in particular as characterized by its oxidative potential, is associated with biomarkers of oxidative stress and inflammation as well as genotoxic effects., Objective: The ultimate goal is to develop exposure reduction strategies based on exposure determinants that best predict MWF-related health outcomes. The following relationships will be explored: (1) exposure determinants and measured exposure; (2) occupational exposure and preclinical and clinical effect markers; (3) exposure biomarkers and biomarkers of effect in both exhaled breath condensate and urine; and (4) biomarkers of effect, genotoxic effects and respiratory symptoms., Methods: At least 90 workers from France and Switzerland (30 controls, 30 exposed to straight MWF and 30 to aqueous MWF) were followed over three consecutive days after a nonexposed period of at least two days. The exposure assessment is based on MWF, metal, aldehyde, and ultrafine particle number concentrations, as well as the intrinsic oxidative potential of aerosols. Furthermore, exposure biomarkers such as metals, metabolites of polycyclic aromatic hydrocarbons and nitrosamine are measured in exhaled breath condensate and urine. Oxidative stress biomarkers (malondialdehyde, 8-isoprostane, 8-hydroxy-2'-deoxyguanosine, nitrates, and nitrites) and exhaled nitric oxide, an airway inflammation marker, are repeatedly measured in exhaled breath condensate and urine. Genotoxic effects are assessed using the buccal micronucleus cytome assay. The statistical analyses will include modelling exposure as a function of exposure determinants, modelling the evolution of the biomarkers of exposure and effect as a function of the measured exposure, and modelling respiratory symptoms and genotoxic effects as a function of the assessed long-term exposure., Results: Data collection, which occurred from January 2018 until June 2019, included 20 companies. At the date of writing, the study included 100 subjects and 29 nonoccupationally exposed controls., Conclusions: This study is unique as it comprises human biological samples, questionnaires, and MWF exposure measurement. The biomarkers collected in our study are all noninvasive and are useful in monitoring MWF exposed workers. The aim is to develop preventative strategies based on exposure determinants related to health outcomes., International Registered Report Identifier (irrid): DERR1-10.2196/13744., (©Nancy B Hopf, Eve Bourgkard, Valérie Demange, Sébastien Hulo, Jean-Jacques Sauvain, Ronan Levilly, Fanny Jeandel, Alain Robert, Yves Guichard, Jacques André Pralong, Nathalie Chérot-Kornobis, Jean-Louis Edmé, Pascal Wild. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 02.08.2019.)

Synthetic amorphous silica nanoparticles (SAS) are among the most widely produced and used nanomaterials, but little is known about their carcinogenic potential. This study aims to evaluate the ability of four different SAS, two precipitated, NM-200 and NM-201, and two pyrogenic, NM-202 and NM-203, to induce the transformation process. For this, we used the recently developed in vitro Bhas 42 cell transformation assay (CTA). The genome of the transgenic Bhas 42 cells contains several copies of the v-Ha-ras gene, making them particularly sensitive to tumor-promoter agents. The Bhas 42 CTA, which includes an initiation assay and a promotion assay, was validated in our laboratory using known soluble carcinogenic substances. Its suitability for particle-type substances was verified by using quartz Min-U-Sil 5 (Min-U-Sil) and diatomaceous earth (DE) microparticles. As expected given their known transforming properties, Min-U-Sil responded positively in the Bhas 42 CTA and DE responded negatively. Transformation assays were performed with SAS at concentrations ranging from 2μg/cm 2 to 80μg/cm 2 . Results showed that all SAS have the capacity to induce transformed foci, interestingly only in the promotion assay, suggesting a mode of action similar to tumor-promoter substances. NM-203 exhibited transforming activity at a lower concentration than the other SAS. In conclusion, this study showed for the first time the transforming potential of different SAS, which act as tumor-promoter substances in the Bhas 42 model of cell transformation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The increasing use of nanomaterials in numerous domains has led to growing concern about their potential toxicological properties, and the potential risk to human health posed by silica nanoparticles remains under debate. Recent studies proposed that these particles could alter gene expression through the modulation of epigenetic marks, and the possible relationship between particle exposure and these mechanisms could represent a critical factor in carcinogenicity. In this study, using the Bhas 42 cell model, we compare the effects of exposure to two transforming particles, a pyrogenic amorphous silica nanoparticle NM-203 to those of the crystalline silica particle Min-U-Sil ® 5. Short-term treatment by Min-U-Sil ® 5 decreased global DNA methylation and increased the expression of the two de novo DNMTs, DNMT3a and DNMT3b. NM-203 treatment affected neither the expression of these enzymes nor DNA methylation. Moreover, modified global histone H4 acetylation status and HDAC protein levels were observed only in the Min-U-Sil ® 5-treated cells. Finally, both types of particle treatment induced strong c-Myc expression in the early stage of cell transformation and this correlated with enrichment in RNA polymerase II as well as histone active marks on its promoter. Lastly, almost all parameters that were modulated in the early stage were restored in transformed cells suggesting their involvement mainly in the first steps of cell transformation.

Crystalline silica particles and asbestos have both been classified as carcinogenic by the International Agency for Research on Cancer (IARC). However, because of the limited data available, amorphous silica was not classifiable. In vitro, the carcinogenic potential of natural crystalline and amorphous silica particles has been revealed by the Syrian Hamster Embryo (SHE) cell transformation assay. On the other hand, the genotoxic potential of those substances has not been investigated in SHE cells. And yet, genotoxicity assays are commonly used for hazard evaluation and they are often used as in vitro assays of reference to predict a possible carcinogenic potential. The main objective of this study was to compare the genotoxic potential and the carcinogenic potential of different crystalline and amorphous silica particles in SHE cells. Three silica samples of different crystallinity were used: natural amorphous silica, partially crystallized silica and quartz silica particles. Their genotoxicity were tested through the in vitro micronucleus assay and the comet assay in SHE, and their carcinogenic potential through the SHE transformation assay. In addition, silica samples were also tested with the same genotoxicity assays in V79 hamster-lung cells, a common in vitro model for particle exposure. Results obtained in the micronucleus and the comet assays show that none of the silica was capable of inducing genotoxic effects in SHE cells and only the amorphous silica induced genotoxic effects in V79 cells. However in the SHE cell transformation assays, the partially crystallized and quartz silica were able to induce morphological cell transformation. Together, these data suggest that, in vitro, the short-term genotoxic assays alone are not sufficient to predict the hazard and the carcinogenic potential of this type of particles; SHE transformation assay appears a more reliable tool for this purpose and should be included in the "in vitro battery assays" for hazard assessment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Synthetic amorphous silica nanomaterials (SAS) are extensively used in food and tire industries. In many industrial processes, SAS may become aerosolized and lead to occupational exposure of workers through inhalation in particular. However, little is known about the in vivo genotoxicity of these particulate materials. To gain insight into the toxicological properties of four SAS (NM-200, NM-201, NM-202, and NM-203), rats are treated with three consecutive intratracheal instillations of 3, 6, or 12 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection (cumulative doses of 9, 18, and 36 mg/kg). Deoxyribonucleic acid (DNA) damage was assessed using erythrocyte micronucleus test and the standard and Fpg-modified comet assays on cells from bronchoalveolar lavage fluid (BALF), lung, blood, spleen, liver, bone marrow, and kidney. Although all of the SAS caused increased dose-dependent changes in lung inflammation as demonstrated by BALF neutrophilia, they did not induce any significant DNA damage. As the amount of SAS reaching the blood stream and subsequently the internal organs is probably to be low following intratracheal instillation, an additional experiment was performed with NM-203. Rats received three consecutive intravenous injections of 5, 10, or 20 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection. Despite the hepatotoxicity, thrombocytopenia, and even animal death induced by this nanomaterial, no significant increase in DNA damage or micronucleus frequency was observed in SAS-exposed animals. It was concluded that under experimental conditions, SAS induced obvious toxic effects but did cause any genotoxicity following intratracheal instillation and intravenous injection., (© 2014 Wiley Periodicals, Inc.)

This study was designed to investigate the modulatory effects of submicron and nanosized iron oxide (Fe(2)O(3)) particles on the ovalbumin (OVA)-induced immune Th2 response in BALB/c mice. Particles were intratracheally administered four times to mice before and during the OVA sensitization period. For each particle type, three different doses, namely 4×100, 4×250 or 4×500 μg/mouse, were used and for each dose, four groups of mice, i.e. group saline solution (1), OVA (2), particles (3), and OVA plus particles (4), were constituted. Mice exposed to OVA alone exhibited an allergic Th2-dominated response with a consistent increase in inflammatory scores, eosinophil numbers, specific IgE levels and IL-4 production. When the mice were exposed to OVA and to high and intermediate doses of iron oxide submicron- or nanoparticles, the OVA-induced allergic response was significantly inhibited, as evidenced by the decrease in eosinophil cell influx and specific IgE levels. However, the low dose (4×100 μg) of submicron particles had no significant effect on the OVA allergic response while the same dose of nanoparticles had an adjuvant effect on the Th2 response to OVA. In conclusion, these data demonstrate that the pulmonary immune response to OVA is a sensitive target for intratracheally instilled particles. Depending on the particle dose and size, the allergic response was suppressed or enhanced., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Potential differences in the toxicological properties of nanosized and non-nanosized particles have been notably pointed out for titanium dioxide (TiO(2)) particles, which are currently widely produced and used in many industrial areas. Nanoparticles of the iron oxides magnetite (Fe(3)O(4)) and hematite (Fe(2)O(3)) also have many industrial applications but their toxicological properties are less documented than those of TiO(2). In the present study, the in vitro cytotoxicity and genotoxicity of commercially available nanosized and microsized anatase TiO(2), rutile TiO(2), Fe(3)O(4), and Fe(2)O(3) particles were compared in Syrian hamster embryo (SHE) cells. Samples were characterized for chemical composition, primary particle size, crystal phase, shape, and specific surface area. In acellular assays, TiO(2) and iron oxide particles were able to generate reactive oxygen species (ROS). At the same mass dose, all nanoparticles produced higher levels of ROS than their microsized counterparts. Measurement of particle size in the SHE culture medium showed that primary nanoparticles and microparticles are present in the form of micrometric agglomerates of highly poly-dispersed size. Uptake of primary particles and agglomerates by SHE exposed for 24 h was observed for all samples. TiO(2) samples were found to be more cytotoxic than iron oxide samples. Concerning primary size effects, anatase TiO(2), rutile TiO(2), and Fe(2)O(3) nanoparticles induced higher cytotoxicity than their microsized counterparts after 72 h of exposure. Over this treatment time, anatase TiO(2) and Fe(2)O(3) nanoparticles also produced more intracellular ROS compared to the microsized particles. However, similar levels of DNA damage were observed in the comet assay after 24 h of exposure to anatase nanoparticles and microparticles. Rutile microparticles were found to induce more DNA damage than the nanosized particles. However, no significant increase in DNA damage was detected from nanosized and microsized iron oxides. None of the samples tested showed significant induction of micronuclei formation after 24 h of exposure. In agreement with previous size-comparison studies, we suggest that in vitro cytotoxicity and genotoxicity induced by metal oxide nanoparticles are not always higher than those induced by their bulk counterparts.

The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres.

DNA phosphate oxygens are sites for alkylation leading to phosphotriester adducts (PTEs). PTEs are reported to be both abundant and persistent and so may serve as long-term markers of genotoxicity. Previously, we reported a 32P-postlabeling assay for the specific detection of PTEs plus identification of nucleosides located 5' to PTEs. Using this, we demonstrated the nonrandom nature of ethyl-PTEs (Et-PTEs) in vivo, these results being suggestive of either the nonrandom formation of Et-PTEs in vivo or sequence specific Et-PTE repair. Presently, we report the further development and validation of the 32P-postlabeling assay, to permit the more straightforward determination of nucleosides 5' to PTEs and, using this, have investigated the long-term persistence of PTEs in vivo. Analysis of liver DNA of mice treated in vivo with N-nitrosodiethylamine reveals an initial decline in the level of Et-PTEs (t1/2<24 h) as well as their nonrandom persistence for the duration of the time course, with approximately 37 and approximately 15% of the initial Et-PTEs remaining 4 and 56 days after treatment, respectively. From this, we conclude that Et-PTEs are suitable as long-term markers of genotoxic exposure and that putative PTE repair is not responsible for their nonrandom manifestation. However, the possibility of active repair contributing to the initial decline of Et-PTEs is considered.

It has been proposed that DNA multiply damaged sites (MDS), where more than one moiety in a local region ( approximately 1 helical turn, 10 bp) of the DNA is damaged, are lesions of enhanced biological significance. However, other than indirect measures, there are few analytical techniques that allow direct detection of MDS in DNA. In the present study we demonstrate the potential of protocols incorporating an exonucleolytic snake venom phosphodiesterase (SVPD) digestion stage to permit the direct detection of certain tandem damage, in which two lesions are immediately adjacent to each other on the same DNA strand. A series of prepared oligonucleotides containing either single or pairs of tetrahydrofuran moieties (F), thymine glycol lesions (T(g)) or methylphosphotriester adducts (Me-PTE) were digested with SVPD and the digests examined by either (32)P-end-labelling or electrospray mass spectrometry. The unambiguous observation of SVPD-resistant 'trimer' species in the digests of oligonucleotides containing adjacent F, T(g) and Me-PTE demonstrates that the SVPD digestion strategy is capable of allowing direct detection of certain tandem damage. Furthermore, in studies to determine the specificity of SVPD in dealing with pairs of lesions on the same strand, it was found mandatory to have the two lesions immediately adjacent to each other in order to generate the trimer species; pairs of lesions separated by as few as one or two normal nucleotides behave principally as single lesions towards SVPD.

Many genotoxic carcinogens react with the sugar-phosphate backbone in DNA to form phosphotriester (PTE) adducts. These lesions are relatively abundant and persistent for some alkylating carcinogens and may therefore serve as useful biomarkers with which to assess genotoxic exposure and potential mutagenic risk. In the present study, we have developed a 32p postlabeling method that permits analysis of total methyl and/or ethyl PTE in DNA at the femtomole level. The technique is based on the inability of all known nucleolytic enzymes to cleave the internucleotide PTE bond. Consequently, complete digestion of alkylated DNA with these nucleases in the presence of an alkaline phosphatase yields PTE-dinucleoside phosphates. These species are then converted to the corresponding dinucleoside phosphates (dNpdNs) by treatment with alkali to permit subsequent 32p labeling. The resulting labeled dinucleotides (32pd-NpdN) are then analyzed by PAGE. Validation of this method has been carried out using a polydeoxythymidylic acid oligonucleotide containing a site-specific methyl PTE. The method has been applied to the in vitro analysis of calf thymus (CT) DNA treated with dimethylsulfate (DMS) or diethylsulfate (DES) and to the analysis of liver DNA from mice treated in vivo with nitrosodiethylamine. In each case, autoradiograms of the polyacrylamide gels showed the anticipated five bands representing the sixteen labeled dinucleotides, with proportional increases observed as the concentrations of DMS or DES used in the in vitro treatment of CT DNA were increased. The identity and frequency of the nucleosides located 5' to the PTE lesions were obtained by nuclease P1 digestion of the gel-isolated 32pdNpdN species and by analysis of the released labeled mononucleotides, 32pdN, by high-performance liquid chromatography with radioactivity detection. Results obtained from CT DNA treated with DMS or DES showed that the frequency of the four detected nucleotides reflected the normal nucleoside content of CT DNA, indicating the random formation of methyl and ethyl PTE adducts in the in vitro modified DNA. However, studies using liver DNA from three strains of mice treated in vivo with nitrosodiethylamine indicated that the frequency of the thymidine and the 2'-deoxyguanosine 5' to the ethyl PTE was significantly different from the corresponding normal nucleoside content. These results are indicative of (a) the nonrandom formation of ethyl PTE in vivo and/or (b) base sequence-specific ethyl PTE repair.

Recent cumulative data have shown that tamoxifen and its metabolites inhibit the activation of cAMP phosphodiesterase by calmodulin (CaM). In this study, the interaction of antiestrogens with CaM was investigated using a hydrophobic fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS). Tamoxifen (TAM) enhanced the fluorescence of TNS bound to CaM and shifted the emission maximum to lower wavelengths. These effects were concentration-dependent. No change in the apparent affinity of TNS for CaM was noted in the presence of TAM. These results suggest that TAM bound to CaM at sites distinct from those of TNS and induced a change in TNS environment. Interaction of TAM metabolites with CaM depended on the degree of alteration of the dimethylaminoethoxy side-chain. Thus, N-desmethylation or N-di-desmethylation notably reduced the interaction of the drug with the macromolecule by 24 and 77% respectively. Side-chain deamination to the primary alcohol (metabolite Y) totally suppressed the interaction. The ability of these different metabolites to interact with CaM correlates with their efficiency to inhibit CaM-dependent cAMP phosphodiesterase and their growth inhibitory potency reported previously.

Immunoaffinity gels able to bind 1,N6-ethenodeoxyadenosine 3'-monophosphate (3'-epsilon dAMP) and 3,N4-ethenodeoxycytidine 3'-monophosphate (3'-epsilon dCMP) were prepared. These gels retained their specific binding capacity for 3'-epsilon dAMP or 3'-epsilon dCMP in the presence of a large excess (> 2 mg per column) of unmodified 3'-deoxynucleotide monophosphates. 3'-epsilon dAMP and 3'-epsilon dCMP were 32P-postlabelled in the presence of [gamma-32P]ATP and T4 polynucleotide kinase to give either 3',5'-[5'-32P]-bisphosphates or 5'-[32P]monophosphates. 3',5'-[5'-32P]Bisphosphates were recovered from the labelling mixture by immunoprecipitation and quantitated by Cerenkov counting (method A). The detection limit of this procedure was 1 fmol with an efficiency of 80% for both ethenonucleotides. Alternatively, 5'-[32P]epsilon dAMP and 5'-[32P]epsilon dCMP were analysed by two-dimensional TLC on PEI cellulose and autoradiography (method B). The detection limit of method B was 50 amol of ethenonucleotide. Methods A and B are complementary and can quantify the formation and repair of 3'-epsilon dAMP and 3'-epsilon dCMP in DNA from rats exposed to a low level of vinyl chloride.

Folliculo-stellar cells are present in the anterior lobe of hypophysis of the monkey Macacus irus. These cells limit strait cavities in which one can observe microvillosites at the apical pole of the cells. The folliculo-stellar cells possess long and thin fibers which insert themselves between different types of granular cells and sometimes extend to the peri-capillar regions. Numerous organelles are present in these folliculo-stellar cells, especially microfilaments.

Antehypophysial somatotropic and prolactinic cells of the Hedgehog have isolated cilia composed by a ring of 9 or 8 double tubules with or without a central tubule; this single cilia is in relation with a basal corpuscule. A granulo-filamentous neck joins the basal corpuscule to the subjacent centriole; from this granulo-filamentous neck accompanying the complex basal corpuscule-centriole, microtubules and striated filaments extend towards the Golgi apparatus. The single cilia, probably of chemoreceptor significance, appears in relation with the Golgi complex, a zone of granule formation.

The anterior lobe of the hedgehog pituitary possess folliculo-stellate cells. These agranular (or few granular) cells, connected together by juctional complexes in relation with mitochondria, limit small cavities; at the luminar surfaces, microvillosities projecting in the lumen are seen. The lumina of cavities contain, or not, an electron dense substance, but not remnants of glandular cells. Long slender cytoplasmic processes, extended between adjacent granular cells, takes contact with the basement parenchymatous membrane, where they form a so-called "vascular foot". The cytoplasm of these cells contains a variety of organelles; their development is variable and appears in relation with the functional activity of the cell.

Clinical, histological, ultrastructural and biochemical studies have been performed in a living 20-month-old infant with GM1-gangliosidosis type 2. Rectum, brain and liver biopsies were done. The histological and ultrastructural examination revealed the presence of cytoplasmic membranous bodies in the nervous system and a vacuolisation of the visceral parenchymatous cells, particularly histiocytes. The diagnosis was established by the finding of a generalized beta-galactosidase deficiency and an accumulation of GM1-ganglioside in brain. In leukocytes, the activity of p-nitrophenyl-beta-galactosidase was below 5%, and that of GM1-ganglioside beta-galactosidase below 1% of values obtained in controls. In cerebral tissue, GM1 ganglioside constituted 80% of total gangliosides; its concentration was 15 times that in age-matched controls. No accumulation of GM1 could be evidence in liver. Enzymatic examination of leukocytes obtained from the consanguineous parents revealed heterozygote values.