4-Hydroxybenzoic acid serves as an endogenous ring precursor for antroquinonol biosynthesis in Antrodia cinnamomea.

Antrodia cinnamomea, an endemic fungus species of Taiwan, has long been used as a luxurious dietary supplement to enhance liver functions and as a remedy for various cancers. Antroquinonol (AQ), identified from the mycelium of A. cinnamomea, is currently in phase II clinical trials in the USA and Taiwan for the treatment of non-small-cell lung cancer. In the previous studies, we have demonstrated that AQ and 4-acetylantroquinonol B (4-AAQB) utilize orsellinic acid, via polyketide pathway, as the ring precursor, and their biosynthetic sequences are similar to those of coenzyme Q. In order to test 4-hydroxybenzoic acid (4-HBA), synthesized via shikimate pathway, is the ring precursor of AQ analogs, the strategy of metabolic labeling with stable isotopes was applied in this study. Here we have confirmed that 4-HBA serves as the ring precursor for AQ but not a precursor of 4-AAQB. Experimental results indicated that A. cinnamomea preferentially utilizes endogenous 4-HBA via shikimate pathway for AQ biosynthesis. Exogenous tyrosine and phenylalanine can be utilized for AQ biosynthesis when shikimate pathway is blocked by glyphosate. The benzoquinone ring of 4-AAQB is synthesized only via polyketide pathway, but that of AQ is synthesized via both polyketide pathway and shikimate pathway. The precursor-products relationships diagram of AQ and 4-AAQB in A. cinnamomea are proposed based on the experimental findings.

Biosynthesis of Antroquinonol and 4-Acetylantroquinonol B via a Polyketide Pathway Using Orsellinic Acid as a Ring Precursor in Antrodia cinnamomea.

Antroquinonol (AQ) and 4-acetylantroquinonol B (4-AAQB), isolated from the mycelium of Antrodia cinnamomea, have a similar chemical backbone to coenzyme Q (CoQ). Based on the postulation that biosynthesis of both AQ and 4-AAQB in A. cinnamomea starts from the polyketide pathway, we cultivated this fungus in a culture medium containing [U-13C]oleic acid, and then we analyzed the crude extracts of the mycelium using UHPLC-MS. We found that AQ and 4-AAQB follow similar biosynthetic sequences as CoQ. Obvious [13C2] fragments on the ring backbone were detected in the mass spectrum for [13C2]AQ, [13C2]4-AAQB, and their [13C2] intermediates found in this study. The orsellinic acid, formed from acetyl-CoA and malonyl-CoA via the polyketide pathway, was found to be a novel benzoquinone ring precursor for AQ and 4-AAQB. The identification of endogenously synthesized farnesylated intermediates allows us to postulate the routes of AQ and 4-AAQB biosynthesis in A. cinnamomea.

Attention Deficit/Hyperactivity Disorder and Urinary Nonylphenol Levels: A Case-Control Study in Taiwanese Children.

OBJECTIVE: Nonylphenol (NP) belongs to the family of endocrine disruptors, and it is widely used in industrial applications and is ubiquitous in daily foods. Animal studies have suggested that NP exposure might promote motor hyperactivity, likely by causing deficits in dopaminergic neurons. However, research assessing NP exposure and epidemiology studies on human populations are limited. The aim of this study was to explore the association between child NP exposure and ADHD while considering particular covariants, such as lead levels and dopamine-related gene variations. METHODS: A case-control study was conducted on patients with clinically diagnosed ADHD; the Swanson, Nolan and Pelham, Fourth Revision (SNAP-IV) questionnaire was used to identify normal controls aged 4-15 years. Participants were examined for urinary NP concentrations, blood lead levels, and select single-nucleotide polymorphisms of two dopamine-related genes (D4 dopamine receptor, DRD4, and dopamine transporter, DAT1). Socio-demographic variables, maternal lifestyle factors during pregnancy and family medical history were obtained using a questionnaire. RESULTS: A total of 97 children with doctor-diagnosed ADHD and 110 normal controls were enrolled. The blood lead levels in both groups were similar (1.57±0.73 vs. 1.73±0.77 µg/dL, p = 0.15). No significant difference in urinary NP concentration was found between the children with ADHD and the control subjects (4.52±3.22 µg/g cr. vs. 4.64±2.95 µg/g cr., p = 0.43). ADHD was significantly more prevalent among males in this study (male to female ratio: 5:1 for the ADHD group and 1.3:1 for the control group, p<0.01). The analysis was repeated after excluding the females, but this had no effect on the association between NP and ADHD. The regression model, including or excluding females, indicated no increased odds of having ADHD in the context of NP exposure after adjusting for covariants. CONCLUSION: This study indicated that NP exposure might not promote ADHD in children, even though children in Taiwan had relatively high levels of NP compared to those reported previously and those in developed nations.

Nonylphenol in pregnant women and their matching fetuses: placental transfer and potential risks of infants.

As the predominant environmental biodegradation product of nonylphenol (NP) ethoxylates and with proven estrogenic effects, NP is formed during the alkylation process of phenols. The purposes of this study were (1) to examine maternal and prenatal exposure to NP in Taiwan, (2) to determine the level of placental protection against NP exposure as well as the level of NP in breast milk, and (3) to assess the potential risk for breastfed newborns exposed to NP through the milk. Thirty pairs of maternal and fetal blood samples, placenta, and breast milk during the 1st and the 3rd months of lactation were collected. External NP exposures of these specimens were then analyzed by using high-performance liquid chromatography coupling with fluorescence detection. Next, the socio-demographics, lifestyle, delivery method, dietary and work history were collected using a questionnaire. In addition, the daily intake of NP from consuming breast milk in the 1st and 3rd months for newborns was studied through deterministic and probabilistic risk assessment methods. The geometric means and geometric standard deviation of NP levels in maternal blood, fetal cord blood, placenta, and breast milk in the 1st and 3rd months were 14.6 (1.7) ng/ml, 18.8 (1.8) ng/ml, 19.8 (1.9) ng/g, 23.5 (3.2) ng/ml, and 57.3 (1.4) ng/ml, respectively. The probabilistic percentiles (50th, 75th, and 95th) of daily intake NP in breast milk were 4.33, 7.79, and 18.39 µg/kg-bw/day in the 1st month, respectively, and were 8.11, 10.78, 16.08 µg/kg-bw/day in the 3rd month, respectively. The probabilistic distributions (5th, 25th, and 50th) of risk for infants aged 1 month old were 0.27, 0.64, and 1.15, respectively, and that for infants aged 3 month old were 0.31, 0.46, and 0.62, respectively. Through repeated exposure from the dietary intake of expectant mothers, fetuses could encounter a high NP exposure level due to transplacental absorption, partitioning between the maternal and fetal compartments. Daily NP intake via breast milk in three month-old babies exceeded the tolerable daily intake (TDI) of 5 µg/kg bw/day indicated a potential risk for Taiwan infants.