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Intramural Papers of the Month

By Robin Arnette
April 2009

Infants Fed Soy Formula Have High Exposure to Isoflavones

Since 25 percent of all infant formula is based on soy protein, and soy has been linked to estrogenic effects in adults, a study was performed to examine isoflavone levels in infants and possible hormone effects. The study was a collaborative effort between scientists at the NIEHS, CDC, FDA and The Children's Hospital of Philadelphia.

A total of 166 infants between birth and one year of age were engaged in a partly cross-sectional/longitudinal pilot study to examine their exposure to three isoflavones - genistein, daidzein and equol - from soy formula, cow milk formula and breast milk. Samples were analyzed using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Saliva, blood and urine samples taken simultaneously demonstrated that genistein and daidzein were undetectable in blood and saliva from children fed breast milk or cow milk formula, while equol was only found in a few urine samples. Urinary concentrations of genistein and daidzein were approximately 500 times higher in soy formula-fed infants than those fed cow milk formula.

Although it was known that infants fed soy formula had a higher exposure to these compounds than did those fed cow milk formula or breast milk, this study was the largest to date and the only one to include measurements in blood, saliva and urine. Future longitudinal studies will examine the physical and developmental effects of estrogen exposure on infants, aged birth through two years, fed soy formula, cow milk formula and breast milk.

Citation: Cao Y, Calafat AM, Doerge DDR, Umbach DM, Bernbaum JC, Twaddle NC, Ye X, Rogan WJ. ( Exit NIEHS 2009. Isoflavones in urine, saliva, and blood of infants: data from a pilot study on the estrogenic activity of soy formula. J Expo Sci Environ Epidemiol 19(2):223-234.

PXR and CAR are Responsible for Bone Loss in Patients Taking Antiepileptic and Antimicrobial Medications

According to researchers at NIEHS, the locking of xenobiotic receptors pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR) to the corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) is the key mechanism to the repression of vitamin D3 activation of the CYP24A1 promoter. Since the activation of the CYP24A1 gene was thought to be responsible for bone mineral density loss that occurs in patients taking antiepileptic and antimicrobial drugs, the research team tested the hypothesis using Pxr(+/+) and Pxr(-/-) mice. This research may provide an opportunity to develop drugs for treating hormone- or vitamin-related diseases such as diabetes and osteoporosis by targeting nuclear xenobiotic receptors.

Vitamin D3, essential for the development and maintenance of calcium in bones, dissociates the SMRT from the CYP24A1 promoter and binds to the vitamin D receptor (VDR) within the vitamin D-response element (VDRE) of the CYP24A1. In the absence of vitamin D3, PXR binds to the VDRE of the CYP24A1 and activates the CYP24A1 gene. When Pxr(+/+) and Pxr(-/-) mice were treated with a PXR-activating drug, only the Pxr(+/+) mice exhibited bone mineral density loss. The data suggest that PXR and CAR can be responsible for the bone loss in patients taking PXR-activating drugs.

Citation: Konno Y, Kodama S, Moore R, Kamiya N, Negishi M. ( Exit NIEHS 2009. Nuclear xenobiotic receptor pregnane X receptor locks corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) onto the CYP24A1 promoter to attenuate vitamin D3 activation. Mol Pharmacol 75(2):265-271.

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The Importance of Pol32 in the Repair of Clustered Lesions

Single-strand break (SSB) and base excision repair (BER) enzymes usually fix damage or breaks in one DNA strand, but clustered damage formed by two or more closely spaced base damages or SSBs on opposing strands can cause double-strand breaks (DSBs). The resulting DSBs pose a unique threat to the genome. In a new study, a research team, comprised of investigators from NIEHS and the University of Pittsburgh Cancer Institute, determined this mechanism using single and double mutants of the budding yeast, Saccharomyces cerevisiae.

Since there were few studies that focused on the repair of clustered lesions in living cells, the researchers developed an in vivo assay that measured such lesions produced by methylmethanesulfonate (MMS) - a DNA damaging agent, - in G1 stationary-phase haploid yeast. DNA Polymerase δ (Pol δ), its subunit Pol32, Rad27/Fen1 nuclease, Cdc9 ligase, and the sliding clamp protein PCNA all interact and must be coordinated to process lesions. pol32Δ and rad27-p single mutants had little effect on the repair of MMS clustered damage, but pol32Δ rad27-p double mutants were unable to repair closely spaced lesions, which resulted in DSBs. However, repair of single-strand damage was not altered.

The findings suggest that clustered lesions are effectively repaired only when repair components are tightly coordinated.

Citation: Ma W, Panduri V, Sterling JF, Van Houten B, Gordenin DA, Resnick MA. ( Exit NIEHS 2009. The transition of closely opposed lesions to double-strand breaks during long-patch base excision repair is prevented by the coordinated action of DNA polymerase delta and Rad27/Fen1. Mol Cell Biol 29(5):1212-1221.

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Protein Kinase Cζ Controls Organic Anion Transporters

The activation of protein kinase C zeta (PKCζ) up-regulates organic anion transporter 1 (OAT1) and OAT3, two proteins that play a crucial role in the removal of small anionic drugs, xenobiotics and their metabolites from the kidney, according to a study by a team of researchers from NIEHS and the Medical University of South Carolina. The investigators also found that protein-protein interactions control these two important renal drug transporters.

To examine the protein-protein interactions that are involved in the OAT-mediated secretion of drugs and toxins, the team employed a yeast two-hybrid assay to screen a human cDNA kidney library to identify proteins that associated with OAT3 in the kidney. PKCζ was one of the 23 putative binding partners identified in the assay. Using a fresh renal tissue preparation, the team was able to show that insulin - an upstream activator of PKCζ - increased PKCζ activity and OAT3-mediated uptake of estrone sulfate (ES). Both effects were abolished by using a PKCζ inhibitor.

Further studies demonstrated that PKCζ modified OAT1 activity as well. Inhibition of PKCζ was also shown to block the increase in drug transport seen in response to epidermal growth factor and the activation of protein kinase A. Future experiments with the other 22 putative binding partners of OAT3 may lead to better understanding of both transporter function and drug pharmacology in the kidney.

Citation: Barros SA, Srimaroeng C, Perry JL, Walden R, Dembla-Rajpal N, Sweet DH, Pritchard JB. ( Exit NIEHS 2009. Activation of protein kinase Czeta increases OAT1 (SLC22A6)- and OAT3 (SLC22A8)-mediated transport. J Biol Chem 284(5):2672-2679.

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