Chronic tarsal conjunctivitis.

BACKGROUND: Toxicity is rarely considered in the differential diagnosis of conjunctivitis, but we present here a new form of toxic conjunctivitis with unusual clinical features. Between 2010 and 2013, a new clinical presentation of chronic conjunctivitis unresponsive to normal treatment was noted within a Primary Care Ophthalmology Service. METHODS: Retrospective review of case records and histopathology results. RESULTS: A total of 55 adult patients, all females, presented with epiphora and stickiness. They did not complain of itch and had had symptoms for an average of 9 months. Clinical examination showed bilateral moderate to severe upper and lower tarsal conjunctival papillary reaction, without corneal or eyelid changes and mild bulbar conjunctival hyperaemia in a third of cases. Biopsies were taken in 15 cases to exclude an atypical infection or lymphoma. Histologically, there was a variable superficial stromal lymphocytic infiltrate, involving the epithelium in more severe cases. The majority of the cells were CD3 positive T-lymphocytes and follicle formation was not noted. The clinical history in all cases included prolonged use of eye make- up and other facial cosmetic products. Clinical symptoms of epiphora settled with topical steroid drops, but the clinical signs of chronic tarsal inflammation persisted until withdrawal of the facial wipes thought to contain the inciting agent, though the exact nature of this remains unclear. CONCLUSION: The presentation, appearances, histological features are consistent with a contact allergen-driven chronic conjunctivitis. Steroid treatment provided good relief of symptoms and patients were advised to avoid potential contact allergens. Management remains difficult. Further research into contact allergies of mucous membranes and identification of its allergens is required.

Urethane and N-nitrosodiethylamine are mutagenic for the Syrian hamster fetus.

Urethane and N-nitrosodiethylamine are soluble environmental carcinogens that initiate tumors transplacentally, but have a mixed history of effectiveness in mutagenesis assays in vitro or in vivo with adult rodents. To test for their transplacental mutagenicity, Syrian hamster fetuses at 12 days in gestation were exposed transplacentally to urethane or N-nitrosodiethylamine at 0.5 or 1.0 mM/kg. The fetal cells were isolated on day 13 of gestation and tested for diphtheria toxin resistance as a mutation marker. Both compounds were significantly mutagenic, at both doses, causing 6- to 20-fold increases in mutations compared with controls. Compared with N-nitrosodiethylamine, urethane was somewhat more effective as a mutagen with a more marked dose-response. These results are consistent with mutagenesis as part of the mechanism of transplacental carcinogenicity of urethane and N-nitrosodiethylamine.

HGPRT mutation induction by N-ethyl-N-nitrosourea as measured by 6-thioguanine resistance is higher in male than in female Syrian hamster fetuses.

BACKGROUND: The consequences of mutations in embryonic and fetal cells are serious and contribute to high prenatal sensitivity to mutagenic agents. An understanding of the factors that influence the yield of such mutations is important for management of adverse effects of perinatal exposures. Resistance to 6-thioguanine (6-TG) can be utilized to study mutational events at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. HGPRT is X-linked and recessive. According to the Lyon hypothesis, male cells have only one X-chromosome and female cells randomly inactivate the second X-chromosome. This leads to the prediction that X-linked genes should be equally sensitive to the mutagenic effects of toxicants in male and female fetuses. METHODS: We tested this supposition by in utero exposure of Syrian hamster fetuses to N-ethyl-N-nitrosourea (ENU) at day 12 of gestation. ENU is a strong carcinogen and mutagen. HGPRT mutations were detected by selection with 6-TG. RESULTS: Surprisingly. the male cells had 4 to 5 times more 6-TG mutants than female cells, in two separate experiments (p<0.001). Ouabain resistance, reflecting a co-dominant autosomal locus, was used as a control, and we found that there was no significant difference between male and female cells (p=0.549). CONCLUSIONS: Possible reasons for the sex difference in mutations include escape of the second X-chromosome from inactivation in some of the female cells, or higher mutability in male cells. In any event, there is a gender difference in vulnerability to mutation of an X-linked gene that has previously not been appreciated, and that may be relevant to toxicological studies of such genes. HGPRT is frequently used to monitor mutagenic events in human fetuses.

Alternate paths from epidermal growth factor receptor to Akt in malignant versus nontransformed lung epithelial cells: ErbB3 versus Gab1.

In many human lung adenocarcinoma cell lines, a pathway involving epidermal growth factor receptor (EGFR), ErbB2 and ErbB3 receptors, phosphatidyl inositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3-beta (GSK3-beta), and cyclin D1 controls cell growth, survival, and invasiveness. We have investigated this pathway in paired transformed/nontransformed cell lines from murine peripheral lung epithelium, E9/E10 and A5/C10. The E9 and A5 carcinoma lines expressed ErbB3 and transforming growth factor-alpha (TGF-alpha) and responded to TGF-alpha stimulation with protein complex formation including the p85 regulatory subunit of PI3K, activation of Akt, phosphorylation of GSK3-beta, and increased cyclin D1 protein and the cell cycle. ErbB3 and TGF-alpha were not detected in the nontransformed E10 and C10 cell lines. Nevertheless, exposure of E10 or C10 cells to TGF-alpha activated PI3K and Akt and increased cyclin D1 and cell growth. The effector pathway from the EGFR to PI3K in these nontransformed cells included the adaptor Grb2, the docking protein Gab1, and the phosphatase Shp2. Gab1 was highly expressed in E10 and C10 cells but not in the malignant E9 and A5 sister lines. Complexes of EGFR/Grb2/Gab1/Shp2 after TGF-alpha stimulation were prominent only in E10 and C10 cells. Thus, alternate pathways downstream of EGFR regulate mitosis in these paired malignant versus nontransformed lung cell lines.

The mutagenic effects of 7,12-dimethylbenz[a]anthacene, 3-methylcholanthrene and benzo[a]pyrene to the developing Syrian hamster fetus measured by an in vivo/in vitro mutation assay.

The transplacental mutagenicity of three polycylic aromatic hydrocarbons, 7,12-dimethylbenz[a]anthacene (DMBA), 3-methylcholanthrene (MC) and benzo[a]pyrene (BP), was measured by an in vivo/in vitro mutation assay. Fetal sensitivity and dose-response characteristics with regard to transplacental mutagenesis by these compounds have never been quantified. In the current experiment, pregnant Syrian hamsters were exposed to these compounds at day 12 of gestation. Twenty-four hours later the fetuses were removed and their cells were allowed a 5-day expression time in culture. They were then seeded for colony formation and also for mutation selection by diphtheria toxin. DMBA at 0.2 mmol/kg (51.3 mg/kg) had an induced mutant frequency of 1.56 x 10(-4) mutants per surviving cell. This was 598 times the historical control. DMBA at 0.2 mmol/kg was 3.6 times more potent than the highly mutagenic positive control, ethylnitrosourea, at 1 mmol/kg. DMBA also caused a dose-dependent increase in cloning efficiency, which was highly correlated with mutation rate. BP and MC were less effective than DMBA, causing increased mutations that were 31.6 and 17.7 times the historical control, respectively, and for neither was there any correlation of mutation rate with cloning efficiency. The special effectiveness of DMBA as a transplacental mutagen may relate to its ability to cause increased cell division and fixation of DNA lesions as mutations.

Cell cycle activation in lung adenocarcinoma cells by the ErbB3/phosphatidylinositol 3-kinase/Akt pathway.

Although ErbB3, a member of the epidermal growth factor receptor family, has been implicated in mammary tumorigenesis, investigation of its role in lung tumorigenesis has been limited. We found that ErbB3 was present at high levels in five of seven human lung adenocarcinoma cell lines examined, along with its ligands, heregulins alpha and beta, whereas ErbB3 was absent from HPL1D, a non- transformed cell line from human pulmonary peripheral epithelium. Interactions and effects of ErbB3 were studied in detail in adenocarcinoma lines H441 and H1373. Complexes containing phosphorylated ErbB2, phosphorylated ErbB3 and the p85 regulatory subunit of phosphoinositidyl 3-kinase were detected by co-immunoprecipitation experiments and were present constitutively even in the absence of serum-stimulated cell division. Serum treatment increased the pErbB3/p85 complexes and also stimulated phosphorylation of Akt and GSK3beta, increase in cyclin D1 and cell cycle progression, and these events were blocked by the Akt activation inhibitor LY294002. An ErbB3-specific antisense oligonucleotide reduced amounts of ErbB3 protein and p85 complex in both cell lines, and significantly suppressed cell proliferation. These results together suggest involvement of ErbB3 in growth of lung adenocarcinomas, through activation of phosphoinositidyl 3 kinase and Akt, inactivation of GSK3beta and stabilization of cyclin D1 for cell cycle maintenance. It could be a useful therapeutic target.

Increased K-ras protein and activity in mouse and human lung epithelial cells at confluence.

Although K-ras is frequently mutated in lung adenocarcinomas, the normal function of K-ras p21 in lung is not known. In two mouse (E10 and C10) and one human (HPL1D) immortalized lung cell lines from peripheral epithelium, we have measured total K-ras p21 and active K-ras p21-GTP during cell proliferation and at growth arrest caused by confluence. In all three cell types, total K-ras p21 increased 2- to 4-fold at confluence, and active K-ras p21-GTP increased 10- to 200-fold. It was estimated that 0.03% of total K-ras p21 was in the active GTP-bound state at 50% confluence, compared with 1.4% at postconfluence. By contrast, stimulation of proliferation by serum-containing medium did not involve K-ras p21 activation, even though a rapid, marked activation of both Erk1/2 and Akt occurred. At confluence, large increases, up to 14-fold, were seen in Grb2/Sos1 complexes, which may activate K-ras p21. In sum, increased protein expression and activity of K-ras p21 are associated with growth arrest, not with proliferation, in mouse and human lung cell lines.

Effects of glucose on cloning efficiency and mutagenesis of fetal rat cells.

In a previous study, treatment of rats with 10% glucose in the drinking water, as fetuses during gestation and for 1.5 months after delivery, significantly enhanced tumor incidence that resulted from N-methyl-N-nitrosourea (MNU, 20 mg/kg) given transplacentally on gestation day 21, with a 1.6-fold increase in overall tumor incidence. We investigated whether glucose would have an effect on MNU-induced mutation in fetal F-344 rat somatic cells as measured in an in vivo/in vitro assay. Rat fetuses were exposed transplacentally to MNU on gestation day 16 and to a 10% glucose solution from gestation day 7 to day 17. Cells were isolated on gestation day 17 for determination of cloning efficiency and for selection of 6-thioguanine (6-TG)-resistant HGPRT mutants. Cloning efficiency of the fetal cells exposed to MNU alone was 22.6+/-2.3% S.E., while that for cells from fetuses exposed to MNU+glucose was 27.5+/-1.6% S.E., which was a significant difference (P=0.018). This indicates an effect of glucose on cell proliferation and survival. MNU treatment significantly increased the mutation frequency of fetal cells from a spontaneous value of 0.4 x 10(-6) per viable cell to (8.8+/-1.8 S.E.,) x 10(-6) (P=0.0087). The coexposure to MNU and glucose yielded a mutant frequency per plate of 0.62+/-0.05 S.E., which was a 1.5-fold increase compared to MNU alone (0.43+/-0.11 S.E., P=0.075. In summary, the data indicate that glucose during pregnancy increases proliferation/survival of fetal cells and possibly also mutation rate.

X-ray induced mutation in Syrian hamster fetal cells.

Transabdominal X-rays are a risk factor for childhood leukemia, and X-ray exposure of mouse fetuses has led to increases in both mutations and initiated tumors in offspring. However, fetal sensitivity and dose-response characteristics with regard to transplacental mutagenesis by X-rays have never been quantified. In the current experiment, pregnant Syrian hamsters at day 12 of gestation were irradiated with 300-kV X-rays. Twenty-four hours later, the fetuses were removed and their cells were allowed a 5 day expression time in culture. They were then seeded for colony formation and also for mutation selection by 6-thioguanine (6-TG). Mutation frequency was linear over the entire dose range, 10-600 R. The average induced 6-TG mutant frequency was 4.7 x 10(-7) per R. These results suggest that fetal cells are highly sensitive to induction of mutations by X-rays, and that a no-effect threshold is not likely. The 10 R dose caused a 25-fold increase in mutation frequency over the historical control, 45 x 10(-7) versus 1.8 x 10(-7), an increase per R of 2.5-fold. Increased risk of childhood cancer related to obstetrical transabdominal X-ray has also been estimated at 2.5-fold per R. Thus, our results are consistent with mutation contributing to this effect.