Jacalyn M. Green

Objectives: To investigate the effects of dietary folate and sex on histopathology of oral squamous cell carcinoma in mice.Materials and Methods: Mice (C57Bl/6, 30/sex) were fed either a deficient folate or sufficient folate diet. Vehicle or 4‐nitroquinoline1‐oxide (50 μg/mL) in vehicle were administered in drinking water for 20 weeks, followed by 6 weeks of regular drinking water. Oral lesions were observed weekly. Tongues were studied for histopathologic changes. Immunohistochemical techniques were used to measure cell proliferation (Ki67+), and to quantify expression of folate receptor, reduced folate carrier, and proton‐coupled folate transporter. T cells, macrophages, and neutrophils were counted and normalized to area.Results: All 4NQO‐treated mice developed oral tumors. Dietary folate level did not affect tumor burden. More tumors were observed on the ventral aspect of the tongue than in other locations within the oral cavity. 4‐nitroquinoline‐1‐oxide‐treated mice displayed 27%–46% significantly lower expression of all three folate transport proteins; diet and sex had no effect on folate transporter expression. T‐cell and neutrophil infiltration in tongues were 9.1‐fold and 18.1‐fold increased in the 4‐nitroquinoline‐1‐oxide‐treated mouse tongues than in controls.Conclusion: Treatment with 4NQO was the primary factor in determining cancer development, decreased folate transport expression, and lymphoid cell infiltration.

Risk factors for liver cancer include tobacco use, alcohol consumption, obesity, and male sex. Administration of 4-nitroquinonline-1-oxide (4NQO) in drinking water mimics the effects of tobacco and leads to oral carcinoma in mice. This study compared the effects of diets high and low in saturated fat (HF and LF, respectively), and sex, on liver histopathology in 4NQO-treated mice and controls. We hypothesized that 4NQO would cause histopathological changes in liver, and that a HF diet would increase hepatic pathology when compared to the LF diet. Mice (C57Bl/6, 36/sex), were divided into a low fat (10 kcal% fat; LF) or high fat (60 kcal% fat, HF) diet. Mice were further subdivided into one of 3 water treatment groups for 17 weeks: water (control), vehicle (1.25% propylene glycol in water [PG]), or 4NQO in (50 μg/ml; 4NQO). All mice were subsequently given water alone for 6 more weeks. Upon euthanasia, livers were harvested, fixed, sectioned, and stained with hematoxylin and eosin (H&E). H&E slides were graded for histopathology; frozen liver samples were analyzed for triglyceride content. Trichrome stained sections were graded for fibrosis. CD3+ T cells, CD68+ macrophages, and Ly6+ neutrophils were detected by immunohistochemistry. Compared to water controls, 4NQO-treatment caused mouse liver histopathological changes such as fibrosis, and increases in hepatic neutrophils, T cells, and macrophages. HF diet exacerbated pathological changes compared to LF diet. Male controls, but not females, demonstrated severe steatosis and increased triglyceride content. 4NQO treatment decreased hepatic fat accumulation, even in animals on a HF diet. In conclusion, this murine model of oral cancer may serve as a model to study the effects of tobacco and diet on liver.

Objective To compare the effects of dietary fat and sex on murine oral squamous cell carcinoma pathology. Materials and methods Male and female C57Bl/6 mice (36/sex) received a low‐fat (10 kcal%) or high‐fat (60 kcal%) diet. Water (control), vehicle, or 4‐nitroquinoline‐1‐oxide in vehicle (50 µg/ml) was provided for 17 weeks followed by 6 additional weeks of water. Oral lesion development was recorded weekly. Histopathologic changes in tongues were examined and T cells (CD3+), macrophages (CD68+), and neutrophils (Ly6+) were quantified. Results All 4‐nitroquinoline‐1‐oxide treated mice developed oral tumors. High‐fat diet exacerbated pathology, demonstrated by an increased final tumor burden (10.9 ± 4.5 vs 7.9 ± 2.5, mm/mouse, p < 0.05; high‐fat diet vs. low‐fat diet, respectively), and a greater histopathology score. When dietary groups were combined, 4‐nitroquinoline‐1‐oxide treated males displayed higher histopathology scores than females (4.2 ± 0.3 vs. 3.6 ± 0.2, respectively, p< 0.05). Lymphoid cell infiltration was greater in the 4‐nitroquinoline‐1‐oxide mouse tongues than controls: T cells (14.0 vs 0.96 cells/mm²), macrophages (3.6 vs 1.8 cells/mm²), and neutrophils (12.0 vs 0.38 cells/mm²). Conclusion High‐fat diet and male sex increased the pathology of 4‐nitroquinoline‐1‐oxide ‐induced oral cancer. Elevated lymphoid cell infiltration contributed to disease pathology.

Supplementation with the synthetic oxidised folate, folic acid is used to prevent neural tube defects and other symptoms of folate deficiency. However, several unanswered questions remain over folic acid efficacy, safety and interactions with gut microbes. Prevention of a development defect caused by folate deficiency in the nematode worm Caenorhabditis elegans requires > 10 fold higher concentrations of folic acid compared to folinic acid, a reduced folate. Here we show that the major route for folic acid to restore normal development is indirect via the Escherichia coli used to feed C. elegans. This route occurs mainly via the E. coli transporter AbgT, which takes up the folic acid breakdown product para-aminobenzoate-glutamate (PABA-glu). We found that folic acid preparations, including a commercial supplement, contain 0.3- 4.0 % of this breakdown product. Previously, we have shown that inhibiting bacterial folate synthesis increases C. elegans lifespan by removing a life-shortening bacterial activity. Here, we show that folic acid restores bacterial folate synthesis and reverses this lifespan increase. It is still to be determined whether this bacterial route increases host folate levels in humans and if there are situations where increased bacterial folate synthesis has negative health complications.

Peer reviewed article authored by (Green JM). Read article or submit your manuscript for publishing.

The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p -aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p -aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB . One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a K _m value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min ⁻¹ mg ⁻¹ . Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

Escherichia coli AbgT was first identified as a structural protein enabling the growth of p -aminobenzoate auxotrophs on exogenous p -aminobenzoyl-glutamate (M. J. Hussein, J. M. Green, and B. P. Nichols, J. Bacteriol. 180:6260-6268, 1998). The abg region includes abgA , abgB , abgT , and ogt ; these genes may be regulated by AbgR, a divergently transcribed LysR-type protein. Wild-type cells transformed with a high-copy-number plasmid encoding abgT demonstrate saturable uptake of p -aminobenzoyl-glutamate ( K _T = 123 μM); control cells expressing vector demonstrate negligible uptake. The addition of metabolic poisons inhibited uptake of p -aminobenzoyl-glutamate, consistent with this process requiring energy. p- Aminobenzoyl-glutamate taken in by cells expressing large amounts of AbgT alone is not rapidly metabolized to a form that is trapped in the cell, as the addition of nonradioactive p- aminobenzoyl-glutamate to these cells results in a rapid loss of intracellular label. The addition of nonradioactive p -aminobenzoate has no effect. The abgA , abgB , and abgAB genes were cloned into the medium-copy-number plasmid pACYC184; p -aminobenzoate auxotrophs transformed with the clone encoding abgAB demonstrated enhanced ability to grow on low levels of p -aminobenzoyl-glutamate. When transformed with complementary plasmids encoding high-copy levels of abgT and medium-copy levels of abgAB , p -aminobenzoate auxotrophs grew on 50 nM p -aminobenzoyl-glutamate. Our data are consistent with a model of p -aminobenzoyl-glutamate utilization in which AbgT catalyzes transport of p -aminobenzoyl-glutamate, followed by cleavage to p -aminobenzoate by a protein composed of subunits encoded by abgA and abgB. While endogenous expression of these genes is very low under the conditions in which we performed our experiments, these genes may be induced by AbgR bound to an unknown molecule. The true physiological role of this region may be related to some molecule similar to p -aminobenzoyl-glutamate, such as a dipeptide.