Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.     

Pseudomonas aeruginosa cytochrome P450 CYP168A1 is a fatty acid hydroxylase that metabolizes arachidonic acid to the vasodilator 19-HETE

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that is highly prevalent in individuals with cystic fibrosis (CF). A major problem in treating CF patients infected with P. aeruginosa is the development of antibiotic resistance. Therefore, the identification of novel P. aeruginosa antibiotic drug targets is of the utmost urgency. The genome of P. aeruginosa contains four putative cytochrome P450 enzymes (CYPs) of unknown function that have never before been characterized. Analogous to some of the CYPs from Mycobacterium tuberculosis, these P. aeruginosa CYPs may be important for growth and colonization of CF patients’ lungs. In this study, we cloned, expressed, and characterized CYP168A1 from P. aeruginosa and identified it as a subterminal fatty acid hydroxylase. Spectral binding data and computational modeling of substrates and inhibitors suggest that CYP168A1 has a large, expansive active site and preferentially binds long chain fatty acids and large hydrophobic inhibitors. Furthermore, metabolic experiments confirm that the enzyme is capable of hydroxylating arachidonic acid, an important inflammatory signaling molecule present in abundance in the CF lung, to 19-hydroxyeicosatetraenoic acid (19-HETE; K_m = 41 μM, V_max = 220 pmol/min/nmol P450), a potent vasodilator, which may play a role in the pathogen’s ability to colonize the lung. Additionally, we found that the in vitro metabolism of arachidonic acid is subject to substrate inhibition and is also inhibited by the presence of the antifungal agent ketoconazole. This study identifies a new metabolic pathway in this important human pathogen that may be of utility in treating P. aeruginosa infections.     

Metabolism of BYZX in Human Liver Microsomes and Cytosol: Identification of the Metabolites and Metabolic Pathways of BYZX

BYZX, [(E)-2-(4-((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one], belongs to a series of novel acetylcholinesterase inhibitors and has been synthesized as a new chemical entity for the treatment of Alzheimer’s disease symptoms. When incubated with human liver microsomes (HLMs), BYZX was rapidly transformed into its metabolites M1, M2, and M3. The chemical structures of these metabolites were identified using liquid chromatography tandem mass spectrometry and nuclear magnetic resonance, which indicated that M1 was an N-desethylated and C = C hydrogenation metabolite of BYZX. M2 and M3 were 2 precursor metabolites, which resulted from the hydrogenation and desethylation of BYZX, respectively. Further studies with chemical inhibitors and human recombinant cytochrome P450s (CYPs), and correlation studies were performed. The results indicated that the N-desethylation of BYZX and M2 was mediated by CYP3A4 and CYP2C8. The reduced form of β-nicotinamide adenine dinucleotide 2′-phosphate was involved in the hydrogenation of BYZX and M3, and this reaction occurred in the HLMs and in the human liver cytosol. The hydrogenation reaction was not inhibited by any chemical inhibitors of CYPs, but it was significantly inhibited by some substrates of α,β-ketoalkene C = C reductases and their inhibitors such as benzylideneacetone, dicoumarol, and indomethacin. Our results suggest that α,β-ketoalkene C = C reductases may play a role in the hydrogenation reaction, but this issue requires further clarification.     

The Cytochrome P450 Enzyme Responsible for the Production of (Z)‐Norendoxifen in vitro

Norendoxifen, an active metabolite of tamoxifen, is a potent aromatase inhibitor. Little information is available regarding production of norendoxifen in vitro. Here, we conducted a series of kinetic and inhibition studies in human liver microsomes (HLMs) and expressed P450s to study the metabolic disposition of norendoxifen. To validate that norendoxifen was the metabolite of endoxifen, metabolites in HLMs incubates of endoxifen were measured using a HPLC/MS/MS method. To further probe the specific isoforms involved in the metabolic route, endoxifen was incubated with recombinant P450s (CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5 and CYP4A11). Formation rates of norendoxifen were evaluated in the absence and presence of P450 isoform specific inhibitors using HLMs. The peak of norendoxifen was found in the incubations consisting of endoxifen, HLMs, and cofactors. The retention times of norendoxifen, endoxifen, and the internal standard (diphenhydramine) were 7.81, 7.97, and 5.86 min, respectively. The K_m (app) and V_max (app) values of norendoxifen formation from endoxifen in HLM was 47.8 μm and 35.39 pmol min⁻¹ mg⁻¹. The apparent hepatic intrinsic clearances of norendoxifen formation were 0.74 μl mg⁻¹ min. CYP3A5 and CYP2D6 were the major enzymes capable of norendoxifen formation from endoxifen with the rates of 0.26 and 0.86 pmol pmol⁻¹ P450 × min. CYP1A2, 3A2, 2C9, and 2C19 also contributed to norendoxifen formation, but the contributions were at least 6‐fold lower. One micromolar ketoconazole (CYP3A inhibitor) showed an inhibitory effect on the rates of norendoxifen formation by 45%, but 1 μm quinidine (CYP2D6 inhibitor) does not show any inhibitory effect. Norendoxifen, metabolism from endoxifen by multiple P450s that including CYP3A5.     

Human Cytochrome P450 17A1 Conformational Selection: MODULATION BY LIGAND AND CYTOCHROME b5*

Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b₅ (b₅). Notably, titration of b₅ to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b_5, providing structural evidence consistent with the reported ability of b₅ to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.     

Dual Effects of Ketoconazole cis-Enantiomers on CYP3A4 in Human Hepatocytes and HepG2 Cells

Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (−)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (−)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, K_i 0.92 µM for testosterone, IC₅₀ 1.46 µM, K_i 2.52 µM for midazolam; (−)-ketoconazole IC₅₀ 0.90 µM, K_i 0.17 µM for testosterone, IC₅₀ 1.04 µM, K_i 1.51 µM for midazolam).     

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio- and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, Δ⁴-androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16α-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio- and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (K_d) values of the binding between CYP154C3 and these steroids were less than 8 μM as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of Δ⁴-androstene-3,17-dione were determined (K_m, 31.9 ± 9.1 μM; k_cat, 181 ± 4.5 s⁻¹). We concluded that CYP154C3 is a steroid D-ring 16α-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16α-specific hydroxylation activity.     

SDS‐PAGE‐free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes

Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS‐PAGE‐free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1‐butyl‐3‐methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D‐SCX‐RPLC‐ESI‐MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.     

Effect of Methamphetamine on Spectral Binding,Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δA_max and K_D of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δA_max (0.004±0.0003 vs. 0.0068±0.0001) and K_D (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δA_max (0.0038±0.0003 vs. 0.0055±0.0003) and K_D (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced K_D in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.     

Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase Cα-dependent NADPH Oxidase Activation

Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O production, but the receptor(s) and signaling mechanism(s) involved are unknown. The effect of Ang II on O is generally attributed to the AT₁ receptor. In some cells, Ang II stimulates protein kinase C (PKC), whose α isoform (PKCα) can activate NADPH oxidase. We hypothesized that in TALs, Ang II stimulates O via AT₁ and PKCα-dependent NADPH oxidase activation. In rat TALs, 1 nm Ang II stimulated O from 0.76 ± 0.17 to 1.97 ± 0.21 nmol/min/mg (p < 0.001). An AT₁ antagonist blocked the stimulatory effect of Ang II on O (0.87 ± 0.25 nmol/min/mg; p < 0.006), whereas an AT₂ antagonist had no effect (2.16 ± 0.133 nmol/min/mg; p < 0.05 versus vehicle). Apocynin, an NADPH oxidase inhibitor, blocked Ang II-stimulated O by 90% (p < 0.01). Ang II failed to stimulate O in TALs from p47^phox−/− mice (p < 0.02). Monitored by fluorescence resonance energy transfer, Ang II increased PKC activity from 0.02 ± 0.03 to 0.13 ± 0.02 arbitrary units (p < 0.03). A general PKC inhibitor, GF109203X, blocked the effect of Ang II on O (1.47 ± 0.21 versus 2.72 ± 0.47 nmol/min/mg with Ang II alone; p < 0.03). A PKCα- and β-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O (0.59 ± 0.15 versus 2.05 ± 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKCα and PKCβ, we used tubules expressing dominant-negative PKCα or -β. In control TALs, Ang II stimulated O by 2.17 ± 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKCα, Ang II failed to stimulate O (change: −0.30 ± 0.27 nmol/min/mg). In tubules expressing dominant-negative PKCβ1, Ang II stimulated O by 2.08 ± 0.69 nmol/min/mg (p < 0.002). We conclude that Ang II stimulates TAL O production via activation of AT₁ receptors and PKCα-dependent NADPH oxidase.     

Contribution of Baicalin on the Plasma Protein Binding Displacement and CYP3A Activity Inhibition to the Pharmacokinetic Changes of Nifedipine in Rats In Vivo and In Vitro

Baicalin purified from the root of Radix scutellariae is widely used in clinical practices. This study aimed to evaluate the effect of baicalin on the pharmacokinetics of nifedipine, a CYP3A probe substrate, in rats in vivo and in vitro. In a randomised, three-period crossover study, significant changes in the pharmacokinetics of nifedipine (2 mg/kg) were observed after treatment with a low (0.225 g/kg) or high (0.45 g/kg) dose of baicalin in rats. In the low- and high-dose groups of baicalin-treated rats, C _max of total nifedipine decreased by 40%±14% (P<0.01) and 65%±14% (P<0.01), AUC_0–∞ decreased by 41%±8% (P<0.01) and 63%±7% (P<0.01), V_d increased by 85%±43% (P<0.01) and 224%±231% (P<0.01), and CL increased by 97%±78% (P<0.01) and 242%±135% (P<0.01), respectively. Plasma protein binding experiments in vivo showed that C _max of unbound nifedipine significantly increased by 25%±19% (P<0.01) and 44%±29% (P<0.01), respectively, and there was a good correlation between the unbound nifedipine (%) and baicalin concentrations (P<0.01). Furthermore, in vitro results revealed that baicalin was a competitive displacer of nifedipine from plasma proteins. In vitro incubation experiments demonstrated that baicalin could also competitively inhibit CYP3A activity in rat liver microsomes in a concentration-dependent manner. In conclusion, the pharmacokinetic changes of nifedipine may be modulated by the inhibitory effects of baicalin on plasma protein binding and CYP3A–mediated metabolism.     

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous,Xenobiotic-Metabolizing Enzyme in Vertebrates

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a ¹⁰T₅₀ (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.     

Maternal Vitamin D Status in Preeclampsia: Seasonal Changes Are Not Influenced by Placental Gene Expression of Vitamin D Metabolizing Enzymes

Preeclampsia, a hypertensive disorder in pregnancy develops in 2–8% of pregnancies worldwide. Winter season and vitamin D deficiency have been associated with its onset.

Objective

To investigate the influence of season on maternal vitamin D status and placental vitamin D metabolism.

Methods

25-OH vitamin D and 1,25-(OH)₂ vitamin D were measured in maternal serum obtained during the winter or summer months from 63 pregnant women at delivery (43 healthy, 20 preeclampsia). In a subgroup, mRNA expression of CYP24A1 (24-hydroxylase), CYP27B1 (1α-hydroxylase) and VDR (vitamin D receptor) were quantified by real time PCR in placental samples of 14 women with normal pregnancies and 13 with preeclampsia.

Results

In patients with preeclampsia,25-OH vitamin D levels were lower, but differed significantly from controls only in summer (18.21±17.1 vs 49.2±29.2 ng/mL, P<0.001), whereas 1,25-(OH)₂ vitamin D levels were significantly lower only in winter (291±217 vs 612.3±455 pmol/mL, P<0.05). A two-factorial analysis of variance produced a statistically significant model (P<0.0001) with an effect of season (P<0.01) and preeclampsia (P = 0.01) on maternal 25-OH vitamin D levels, as well as a significant interaction between the two variables (P = 0.02). Placental gene expression of CYP24A1, CYP27B1, and VDR did not differ between groups or seasons. A negative correlation between placental gene expression of CYP24A1 and CYP27B1 was observed only in healthy controls (r = −0.81, P<0.0001).

Summary

Patients with preeclampsia displayed lower vitamin D serum levels in response to seasonal changes.The regulation of placental CYP24A1, but not of the VDR or CYP27B1 might be altered in preeclampsia.     

Auto-Induction Effect of Chloroxoquinoline on the Cytochrome P450 Enzymes of Rats Associated with CYP 3A and 1A

To investigate the auto-induction of cytochrome P450 (CYP450) by Chloroxoquinoline (CXL), a novel anticancer drug. Three experiments related to the induction of CYP450 were performed: a) In vitro use of the rat fresh hepatocytes model; b) In vivo ‘cocktail’ of CYP450 probe model; c) Pharmacokinetic (PK) study of the single and multiple doses. Some typical CYP enzyme probes and inducers were used in these experiments and were all determined by HPLC-MS/MS. The expression levels of CYP3A and CYP1A mRNA were analyzed by the real time polymerase chain reaction (RT-PCR) technique. The PK studies showed that the area under the curve (AUC_0-t) and the peak concentration (C_max) of the multiple doses were approximately 2.4-fold and 1.9-fold lower than those of the single dose, respectively (p< 0.05). Subsequent studies were conducted to study the possible induction of CXL on CYP 450. The in vivo ‘cocktail’ administration of CYP450 probe model indicated that 5 d pretreatment with CXL resulted in a mean 4.6 times increase in the metabolites/probe plasma ratios for CYP 3A and a 336% increase for CYP 1A than those of the negative control (p< 0.05). The induction effect of CXL on CYP450 was further evaluated on rat hepatocytes with four concentrations (1, 10, 50 and 100 μmol/L). Compared with the negative control, the mRNA levels of CYP 1A2 increased significantly in rat hepatocytes after treatment with 10, 50 and 100 μmol/L CXL (p< 0.05). While significant inductions of CYP 3A1 were observed in the entire treated groups. The results of the present study demonstrate enhanced and induced expression of CYP 3A and CYP 1A in response to CXL exposure in rats, suggesting that CXL is an auto-inducer of CYP 3A and CYP 1A.     

Lactobacillus rhamnosus Strain GG Reduces Aflatoxin B1 Transport, Metabolism, and Toxicity in Caco-2 Cells

The probiotic Lactobacillus rhamnosus GG is able to bind the potent hepatocarcinogen aflatoxin B₁ (AFB₁) and thus potentially restrict its rapid absorption from the intestine. In this study we investigated the potential of GG to reduce AFB₁ availability in vitro in Caco-2 cells adapted to express cytochrome P-450 (CYP) 3A4, such that both transport and toxicity could be assessed. Caco-2 cells were grown as confluent monolayers on transmembrane filters for 21 days prior to all studies. AFB₁ levels in culture medium were measured by high-performance liquid chromatography. In CYP 3A4-induced monolayers, AFB₁ transport from the apical to the basolateral chamber was reduced from 11.1% ± 1.9% to 6.4% ± 2.5% (P = 0.019) and to 3.3% ± 1.8% (P = 0.002) within the first hour in monolayers coincubated with GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml, respectively). GG (1 × 10¹⁰ and 5 × 10¹⁰ CFU/ml) bound 40.1% ± 8.3% and 61.0% ± 6.0% of added AFB₁ after 1 h, respectively. AFB₁ caused significant reductions of 30.1% (P = 0.01), 49.4% (P = 0.004), and 64.4% (P < 0.001) in transepithelial resistance after 24, 48, and 72 h, respectively. Coincubation with 1 × 10¹⁰ CFU/ml GG after 24 h protected against AFB₁-induced reductions in transepithelial resistance at both 24 h (P = 0.002) and 48 h (P = 0.04). DNA fragmentation was apparent in cells treated only with AFB₁ cells but not in cells coincubated with either 1 × 10¹⁰ or 5 × 10¹⁰ CFU/ml GG. GG reduced AFB₁ uptake and protected against both membrane and DNA damage in the Caco-2 model. These data are suggestive of a beneficial role of GG against dietary exposure to aflatoxin.