Effects of bisphenol A on murine salivary glands and human tumor cell lines.

Bisphenol A (BPA) is an endocrine-disrupting chemical with a potential role in endocrine cancers. However, the effects of BPA on the salivary glands have been barely explored. We investigated the impact of in vivo sub-chronic exposure to BPA and its in vitro effects on human salivary gland mucoepidermoid carcinoma cell lines. Male and female mice were exposed to BPA (30 mg/kg/day). Sublingual and submandibular salivary glands from an estrogen-deficiency model were also analyzed. BPA concentration in salivary glands was evaluated by gas chromatography coupled to ion trap mass spectrometry. Immunohistochemical analysis using anti-p63 and anti-α-SMA antibodies was performed on mouse salivary gland tissues. Gene expression of estrogen receptors alpha and beta, P63 and α-SMA was quantified in mouse salivary gland and/or mucoepidermoid (UM-HMC-1 and UM-HMC-3A) cell lines. Cell viability, p63 and Ki-67 immunostaining were evaluated in vitro. BPA disrupted the tissue architecture of the submandibular and sublingual glands, particularly in female mice, and increased the expression of estrogen receptors and p63, effects that were accompanied by significant BPA accumulation in these tissues. Conversely, ovariectomy slightly impacted BPA-induced morphological changes. In vitro, BPA did not affect the proliferation of neoplastic cells, but augmented the expression of p63 and estrogen receptors. The present data highlight a potential harmful effect of BPA on salivary gland tissues, particularly in female mice, and salivary gland tumor cells. Our findings suggest that estrogen-dependent pathways may orchestrate the effects of BPA in salivary glands.

Agent Orange exposure and cancer incidence in Korean Vietnam veterans: a prospective cohort study.

BACKGROUND: During the Vietnam War, US and allied military sprayed approximately 77 million liters of tactical herbicides including Agent Orange, contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. To the authors' knowledge, few studies to date have examined the association between Agent Orange exposure and cancer incidence among Korean veterans who were exposed to Agent Orange during the Vietnam War. METHODS: An Agent Orange exposure index, based on the proximity of the veteran's military unit to the area that was sprayed with Agent Orange, was developed using a geographic information system-based model. Cancer incidence was followed for 180,251 Vietnam veterans from 1992 through 2003. RESULTS: After adjustment for age and military rank, high exposure to Agent Orange was found to significantly increase the risk of all cancers combined (adjusted hazards ratio [aHR], 1.08). Risks for cancers of the mouth (aHR, 2.54), salivary glands (aHR, 6.96), stomach (aHR, 1.14), and small intestine (aHR, 2.30) were found to be significantly higher in the high-exposure group compared with the low-exposure group. Risks for cancers of all sites combined (aHR, 1.02) and for cancers of the salivary glands (aHR, 1.47), stomach (aHR, 1.03), small intestine (aHR, 1.24), and liver (aHR, 1.02) were elevated with a 1-unit increase in the exposure index. CONCLUSIONS: Exposure to Agent Orange several decades earlier may increase the risk of cancers in all sites combined, as well as several specific cancers, among Korean veterans of the Vietnam War, including some cancers that were not found to be clearly associated with exposure to Agent Orange in previous cohort studies primarily based on Western populations.

Nivolumab plus ipilimumab in advanced salivary gland cancer: a phase 2 trial.

Salivary gland cancers (SGCs) are rare, aggressive cancers without effective treatments when metastasized. We conducted a phase 2 trial evaluating nivolumab (nivo, anti-PD-1) and ipilimumab (ipi, anti-CTLA-4) in 64 patients with metastatic SGC enrolled in two histology-based cohorts (32 patients each): adenoid cystic carcinoma (ACC; cohort 1) and other SGCs (cohort 2). The primary efficacy endpoint (≥4 objective responses) was met in cohort 2 (5/32, 16%) but not in cohort 1 (2/32, 6%). Treatment safety/tolerability and progression-free survival (PFS) were secondary endpoints. Treatment-related adverse events grade ≥3 occurred in 24 of 64 (38%) patients across both cohorts, and median PFS was 4.4 months (95% confidence interval (CI): 2.4, 8.3) and 2.2 months (95% CI: 1.8, 5.3) for cohorts 1 and 2, respectively. We present whole-exome, RNA and T cell receptor (TCR) sequencing data from pre-treatment and on-treatment tumors and immune cell flow cytometry and TCR sequencing from peripheral blood at serial timepoints. Responding tumors universally demonstrated clonal expansion of pre-existing T cells and mutational contraction. Responding ACCs harbored neoantigens, including fusion-derived neoepitopes, that induced T cell responses ex vivo. This study shows that nivo+ipi has limited efficacy in ACC, albeit with infrequent, exceptional responses, and that it could be promising for non-ACC SGCs, particularly salivary duct carcinomas. ClinicalTrials.gov identifier: NCT03172624 .

Occurrence of salivary gland tumours in two patients treated with biological agents.

We report two cases of salivary gland tumors arising in two psoriatic patients treated with an anti- TNF-alpha agent. A clear causal relationship could not be established, but the exceptional onset of a bilateral Warthin's tumor in one of these patients should be emphasized.

Subchronic toxicity and genotoxicity of diiodomethyl-p-tolylsulfone (DIMPTS) in laboratory animals.

These studies were conducted to determine subchronic toxicity and genotoxicity of the biocide diiodomethyl-p-tolysulfone (DIMPTS) in rats and dogs. Male and female Sprague-Dawley rats and Beagle dogs were administered DIMPTS for 90-days via the diet at 0, 5, 20, and 80 mg/kg/day to rats and via capsules at 0, 2, 10, and 60 mg/kg/day to dogs. In rats, the only treatment-related finding was squamous metaplasia of the salivary gland duct in the 80 mg/kg/day group. In dogs, female body weights in the high-dose group were significantly lower than controls. Altered clinical pathology parameters were considered secondary to inflammatory changes observed in some of the dogs. Treatment-related alterations were found in the thyroid glands, salivary glands, GI-tract in the mid- and/or high-dose groups. DIMPTS was negative in the four in vitro and one in vivo genotoxicity assays. The toxicological effects noted in the two mammalian species are consistent with the principal toxic effects of iodine, and are proposed to arise from release of iodide from the DIMPTS molecule with toxic sequelae.

A Single-Arm, Prospective, Phase II Study of Cisplatin Plus Weekly Docetaxel as First-Line Therapy in Patients with Metastatic or Recurrent Salivary Gland Cancer.

PURPOSE: Salivary gland cancers (SGCs) are relatively rare but comprise various histologic subtypes, which complicates design of prospective trials. Systemic chemotherapy plays a limited role in treatment of SGCs, but cisplatin and docetaxel showed efficacy in a previous preclinical study. Here, we conduct a prospective, phase II study to evaluate the efficacy and toxicities of cisplatin plus weekly docetaxel in patients with metastatic or recurrent SGC. MATERIALS AND METHODS: We included patients with histologically confirmed SGCs of the following subtypes: mucoepidermoid carcinoma, adenocarcinoma, ductal carcinoma, or adenoid cystic carcinoma. Patients had no prior systemic chemotherapy for metastatic or recurrent tumors and at least one measurable lesion. Patients were treated with docetaxel 35 mg/m2 (D1, 8) and cisplatin 70 mg/m2 (D1) every 21 days. RESULTS: Forty-one patients were enrolled between April 2014 and October 2020. The median age was 58 years (range, 32 to 73 years). The most common histologic subtype was adenoid cystic carcinoma (63.4%), followed by ductal carcinoma (24.4%). The most common metastatic site was the lung (75.6%). The median treatment cycle was 5.5 (range, 3 to 8), and the objective response rate was 46.3%, with three complete responses. The median duration of response was 6.8 months (interquartile range, 4.0 to 10.2). The progression-free survival and overall survival were 9.4 months (95% confidence interval [CI], 8.4 to 10.5) and 28.2 months (95% CI, 22.7 to 33.6), respectively. There were no treatment-related deaths. The most common grade 3/4 adverse events were neutropenia (4.9%) and fatigue (4.9%). CONCLUSION: Cisplatin plus weekly docetaxel is effective and tolerable with manageable toxicity as first-line therapy in patients with metastatic or recurrent SGC.

Recurrent bronchial epithelial-myoepithelial carcinoma after local therapy.

We present a rare case of recurrent multiple lesions of bronchial epithelial-myoepithelial carcinoma in a 74-year-old man treated with local resection. Two cellular types were found: epithelial cells and myoepithelial cells. The patient remains asymptomatic at 4-years of follow-up, supporting the fact that epithelial-myoepithelial carcinoma is a tumor of low-grade malignancy.

Influence of diets varying in lipid and protein content on the histopathologic variety of murine salivary gland tumors induced by 9,10-dimethyl-1,2-benzanthracene (DMBA).

The purpose of this study was to analyze the influence of diets varying in lipids and proteins on the histopathologic variety of murine salivary tumors induced by DMBA. 117 BALB/c mice were assigned to experiments one (E1: lipids, males) and two (E2: proteins, males and females), E1 comprising Soy oil (SO); Corn oil (CO, control); Fish oil (FO) and Olein (O) groups and E2, soy protein (SP) and casein (C) groups. Tumors were induced by DMBA and the animals were sacrificed at week 13- post-induction. Tumor volume was calculated. Tumor sections were stained with H-E for histopathologic evaluation. No significant association was found between tumor volume and dietary condition (p > 0.05). In E1, FO animals developed mainly carcinomas (C) (58.8%), the sarcomas (S) and carcinosarcomas (CS) being especially of high-grade type (tumors < 600 mm3). In E2, SP animals developed mainly C (55.6%). Although no significantly different (p > 0.05), S and C were more frequent in female and male mice, respectively. In both E1 and E2, the biggest tumors (> 600 mm3) were mainly high-grade S (87.5%-80%). Dietary fat and soy protein appear to influence the tumor histopathology and thus its prognosis.

Effects of isoproterenol on mammary gland tumors induced by N-nitroso-N-methylurea and salivary gland tumors induced by 7,12-dimethylbenz[a]anthracene.

The effect of isoproterenol (IPR) (20 mg/kg, twice per wk) on mammary gland tumors induced by N-nitroso-N-methylurea (NMU) (40 or 77 mg/kg, iv) was studied. Within 18 weeks 50--60% of the noninbred female Sprague-Dawley rats that received a single injection of NMU developed carcinomas of the mammary gland. In addition, a malignant lymphoma was observed. There was no change in the incidence of tumors if NMU was administered at the time that DNA synthesis was stimulated in the submandibular glands by two injections of IPR (160 mg/kg, ip) given 24 hours apart. Tumors of unequivocal salivary gland origin were not observed, irrespective of whether NMU was given at the peak of stimulated DNA synthesis or without pretreatment with IPR. However, in 10% of the tumor-bearing rats, tumors were found in the neck region. These tumors could be separated from the salivary glands by dissection and were of mammary gland origin. Chronic treatment with IPR caused marked enlargements of the parotid and submandibular glands with hypertrophy of the acinar cells and degeneration of the duct system. Such a treatment caused a high death rate but no change in the incidence of the tumors. The effect of chronic IPR treatment (10 mg/kg twice per wk or 5 mg/kg three times per wk) on submandibular gland tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) was also studied. With the intraglandular injection of 100 or 200 micrograms of DMBA, the incidences of squamous cell carcinomas in the glands were 6.8 and 38.6%, respectively. Chronic administration of IPR after the instillation of DMBA caused a high death rate but no statistically significant change in the incidence of salivary gland tumors.

Carcinogenicity of 5-nitrofurans and related compounds with amino-heterocyclic substituents.

Carcinogenicity of eight 5-nitrofurans with heterocyclic substituents at the 2-position of the furan ring was investigated by feeding the chemicals to Sprague-Dawley female rats. N-[5-(5-nitro-2-furyl)-1,3,4-thiadiazol-2-yl]acetamide induced in 30 rats the highest incidence of tumors with the greatest number of tissues involved: forestomach squamous cell tumors (22), kidney pelvis transitional cell carcinomas (15), pulmonary alveolar cell carcinomas (16), hemangioendothelialsarcomas (20) of the intestine, mesentery, liver, lung, and pancreas, and a few tumors of other tissues. 2-Amino-5-(5-nitro-2-furyl)-1,3,4-thiadiazole, 2-amino-5-(5-nitro-2furyl)-1,3,4-oxadiazole, and trans-2-[dimethylamino)methylimino]-5-[2-(5-nitro-2-furyl)vinyl]-1,3,4-oxadiazole produced high incidences of mammary tumors. The other four 5-nitrofurans tested: N-[4-(5-NITRO-2-FURYL)-2-THIAZOLYL]ACETAMIDE;2,3,4-TRIFLUORO-N-[4-(5-NITRO-2-furyl)-2-thiazoly]acetamide;5-(5-nitro-2-furyl)-1,3,4-oxadiazol-2-ol; and N-( [3-(5-nitro-2-furyl)-1,2,4-oxadiazol-5-yl]methyl)acetamide were associated with tumor incidences of 40-60%. Two other chemicals were also tested: 2-Amino-5-nitrothiazole caused a low incidence of breast and kidney pelvis tumors, and 2-amino-4-(p-nitrophenyl)thiazole induced a high incidence of breast and salivary gland adenocarcinomas and lymphomas.

Effect of intestinal microflora and dietary fat on 3,2'-dimethyl-4-aminobiphenyl-induced colon carcinogenesis in F344 rats.

The effect of dietary fat and intestinal microflora on colon and small intestinal carcinogenesis was studied in weanling male F344 germfree and conventional rats fed semipurified diets containing low and high fat. At 7 weeks of age, all animals, except vehicle-treated groups, received weekly s.c. injections of 50 mg 3,2'-dimethyl-4-aminobiphenyl (DMAB) per kg body weight for 20 weeks. The DMAB- and vehicle-treated germfree and conventional rats fed high- and low-fat diets were autopsied 20 weeks after the last injection of DMAB. The incidence of colon, small intestinal, ear duct, and skin tumors was lower in germfree rats than in the conventional animals. The incidence and multiplicity of DMAB-induced colon tumors were higher in conventional rats fed the high-fat diet than in the conventional animals fed the low-fat diet; however, the tumor incidence did not differ significantly between the high- and low-fat-fed germfree animals. Dietary fat had no effect on the incidence of small intestinal tumors in both germfree and conventional rats.

A classification of transplantable tumors in Nb rats controlled by estrogen from dormancy to autonomy.

Transplantable tumor lines were previously established from a variety of estrogen-induced tumors in Nb rats, including tumors of the adrenal cervix, salivary gland, and pancreas, a lymphoma, and a liposarcoma. Spontaneous tumors, however, were found to arise in untreated females and showed the same characteristics. Tumor growth was dependent upon or influenced by estrogen when assessed in estrogenized and unconditioned hosts. Intermitten estrogenization was effective, but tumor growth took place more slowly. The type of response observed led to a new classification of five types of hormone-responsive tumors including tumors inhibited by estrogen. Estrogen-dependent tumor cells might remain dormant indefinitely and not grow in unconditioned animals until stimulated to grow by estrogen. The growth rate of hormone-dependent adrenal carcinomas was related to the amount of estrogen. Tumor growth started more rapidly in the presence of low estrogen dose levels in old rats used as hosts than it did in young rats. Breast carcinomas required the largest amount of estrogen for growth, whereas ovarian thecomas would grow in normal females but not in males. The growth rate in conditioned hosts of most transplanted tumors (some have maintained hormone dependency over 10 years) increased over successive generations. Progression, however, towards a more autonomous state after repeated transplantations was remarkably slow, and a sudden change to autonomy was rarely noted. In contrast, transplants of 9,12-dimethylbenz(a)anthracene-induced mammary carcinomas progressed rapidly to autonomy. Fould's concept of progression (2, 3) has been discussed but the described classification of tumors under hormone influence apparently allows a more detailed analysis of definition of different types of progression.

Tumor induction by a single subcutaneous injection of sterigmatocystin in newborn mice.

Sterigmatocystin, a mycotoxin produced by Aspergillus versicolor, Aspergillus sydowi, Aspergillus nidulans, and a species of Bipolaris, was given to newborn BALB/c X DBA/2F1 (hereafter referred to as CD2F1) mice by a single s.c. administration in 1% gelatin suspension. In an acute toxicity study, the maximum tolerated dose of sterigmatocystin was 5 mug/g body weight. In a chronic study, a single s.c. injection of 5, 1, or 0.5 mug/g body weight gave rise to high incidences of lung and liver adenomas when the animals were killed at the end of 1 year. The incidence of both tumors in mice at the dose of 5 mug/g body weight was statistically significant, and the incidences of lung tumor in female mice and of liver tumor in male mice at the dose of 1 mug/g body weight were also statistically significant, compared with tumors in control mice. Other tumors also were induced in treated mice (two malignant lymphomas and one adenoma of the submaxillary gland), in contrast to a zero incidence in vehicle control mice. These results confirm that a small quantity of sterigmatocystin induces tumors of lung and liver and that the dose of sterigmatocystin is related to the incidence of tumors in mice.

Spontaneous and estrogen-produced tumors in Nb rats and their behavior after transplantation.

Tumors in rats of the Nb strain, arising either spontaneously or after prolonged treatment with s.c. pellets of estrogen, were transplanted to establish whether hormone conditioning was required for their growth. Whereas all spontaneous tumors arising in males and many of those in females were autonomous on transplant, most of those arising in estrogenized rats continued to require hormones for growth after transplantation. The latter included carcinomas of the adrenal cortex, breast, pituitary ectopic tissue, ovary (thecomas), Leydig cells of testis, thymus, pancreas,salivary glands, oribital gland (fibroadenoma), liposarcoma, and lymphoma. Many of the tissues of origin of the tumors have not been considered to be under theinfluence of estrogens. A type of hormone-responsive tumor that was inhibited by estrogen and that grew only in normal rats is described. Ali estrogens tested, including estriol , were interchangeable in action. The incidence of the more common tumors of the adrenal, breast, and pituitary was very low in normal rats, but higher in females. All tumors were more common after estrogenization in both sexes, particularyly in older animals. The secretion of steroids and pitiutary hormones by many tumors led to obvious biological effects. Pituitary secretion led to severe lesions frequently associated with diseases in humans, but the signs of such diseases in the rat apparently were hormone dependent and disappeared if the tumor was removed. The overall results raised the possiblity that estrogens were not carcinogenic per se but stimulated the growth of previously altered cells and that, following their transplantation, this hormone requirement was retained. Irrespective of the mechanism of carcinogenesis, hormone-dependent tumor growth was not irreversible but was controlled in an unexpectedly wide spectrum of organs by exogenous estrogen. Host factors may play a major role in controlling the growth of many tumors and the ultimate course of the disease.

Effects of soy oil on murine salivary tumorigenesis.

Dietary fat influences dimethylbenzanthracene (DMBA)-induced tumorigenesis of several organs, including the salivary glands. There is not enough evidence to suggest that soy oil could also affect growth of salivary tumors. The main purpose of this work therefore was to study the effects of dietary soy oil on macroscopic parameters of chemically induced murine salivary gland tumors. Eighty BALB/c male mice were assigned to four groups: soy oil (SO), corn oil (CO, control), fish oil (FO) and olein (O). Two weeks later, tumors were induced by 9,10-dimethyl-1,2-benzanthracene (DMBA). At the 13th post-injection week, the animals were sacrificed. In vivo tumor diameter, gland volume (total resected mass), tumor volume (microscopically measured), tumor remission and tumor histopathology were analyzed. The initial in vivo tumor diameter, gland and tumor volume were significantly greater in soy oil than in fish oil group. 26.7% of animals on the soy oil diet showed tumor remission. Sarcomas were more often found in the SO group, carcinomas in FO and the mixed-type tumors both in SO and CO groups. This study shows that the soy oil treatment resulted in larger tumors, some of which later became undetectable. It is necessary to further investigate these divergent results.

Intranuclear rodlets in undifferentiated carcinomas of salivary glands in strain A mice in a study involving a tobacco specific nitrosamine, N-nitrosonornicotine.

Undifferentiated carcinomas of the salivary glands were found in 2 of 44 (4.5%) strain A mice injected intraperitoneally with a tobacco specific nitrosamine, N-nitrosonornicotine (NNN). The presence of intranuclear rodlets (INR) in the salivary carcinomas provided the first demonstration of such structures in a non-neuronal tumor in mice. Two types of rodlets were exhibited; one was composed of fibrillar filaments arranged in bundles, and the other was much thicker, branching in form. These INR appeared to be closely associated with nuclear chromatin or nucleoli.

Skin and salivary gland carcinogenicity of 7,12-dimethylbenz[a]anthracene is equivalent in the presence or absence of aryl hydrocarbon receptor.

7,12-Dimethylbenz[a]anthracene (DMBA) is a well-known polycyclic aromatic hydrocarbon (PAH) that causes a variety of tumors in exposed animals. Although PAH carcinogenicity is primarily mediated by the aryl hydrocarbon receptor (AhR) through induction of P450, it is not precisely determined whether AhR regulates the DMBA carcinogenesis in vivo. In this context, we examined the frequency of DMBA-induced tumors and the expressions of mRNAs of P450-CYP1 subfamily and microsomal epoxide hydrolase (mEH) in the skin and submandibular gland using AhR-deficient mice. After DMBA exposure, AhR-/- and AhR+/+ mice showed the same tumor incidences and latency. CYP1A1 was absent in these tissues but was slightly induced in DMBA-treated AhR+/+ mice. In AhR-/- and AhR+/+ mice, constitutive expression of CYP1B1 was evident at equivalent levels, whereas CYP1A2 was not detectable, irrespective of DMBA treatment. mEH was expressed in both tissues of all animals. Collectively, the constitutive levels of CYP1B1 and mEH in the skin and submandibular gland maintain DMBA response in these tissues of AhR-/- mice.

Emergence of osteoblast-like cells in a neoplastic human salivary cancer cell line after treatment with 22-oxa-1alpha, 25-dihydroxyvitamin D3.

A neoplastic clonal cell line, which was prepared by 5-azacytidine treatment of a neoplastic human salivary intercalated duct cell line, was cultivated in the presence of 22-oxa-1alpha, 25-dihydroxyvitamin D3 and 3 mM beta-glycerophosphate. Major alterations, such as expression of type I collagen and alkaline phosphatase as well as of human osteopontin and osteonectin, were observed in these cells with a phenotype similar to osteoblasts. In addition, formation of bone nodule was observed in the cultured cells. The tumors produced by transplantation into nude mice of the clonal cells were treated with 22-oxa-1alpha, 25-dihydroxyvitamin D3 and examined for tumor growth and morphology. Consequently, growth of the treated tumor was significantly suppressed. Moreover, it was found that bone formation was induced in the treated tumor, in which the tumor cells around bone formation expressed human osteopontin and osteonectin mRNA as could be detected by in situ hybridization. The above findings indicate that the emergence of osteoblast-like cells in the human salivary cancer cells occurs in the presence of 22-oxa-1alpha, 25-dihydroxyvitamin D3 and beta-glycerophosphate.

Experimental carcinogenesis in duct-artery ligated rat submandibular gland.

In an attempt to shorten tumor induction time and alter tumor anaplasticity, ductal metaplasia was induced in the submandibular gland of 30 rats by ligation of the duct and artery followed by the implanation of 4-mg pellets of pure DMBA. Control groups consisted of 19-ligated and 15-DMBA implanted rats. The ligate controls showed degeneration and atrophy of the affected gland and replacement of glandular tissue by scar tissue. The DMBA controls followed the expected pattern, with all rats eventually developing well-differentiated squamous cell carcinoma. The CMBA-ligated rats showed a combination of the two processes, each acting separately from the other. There was no difference either in induction time or tumor anaplasticity between the DMBA control rats and the DMBA-ligated rats. It seems that only factors relating to a change in the immunologic response of the host affect the latent period of tumor induction time in submandibular gland carcinogenesis.