A self-assembling peptide matrix used to control stiffness and binding site density supports the formation of microvascular networks in three dimensions.

A three-dimensional (3-D) cell culture system that allows control of both substrate stiffness and integrin binding density was created and characterized. This system consisted of two self-assembling peptide (SAP) sequences that were mixed in different ratios to achieve the desired gel stiffness and adhesiveness. The specific peptides used were KFE ((acetyl)-FKFEFKFE-CONH2), which has previously been reported not to support cell adhesion or MVN formation, and KFE-RGD ((acetyl)-GRGDSP-GG-FKFEFKFE-CONH2), which is a similar sequence that incorporates the RGD integrin binding site. Storage modulus for these gels ranged from ∼60 to 6000Pa, depending on their composition and concentration. Atomic force microscopy revealed ECM-like fiber microarchitecture of gels consisting of both pure KFE and pure KFE-RGD as well as mixtures of the two peptides. This system was used to study the contributions of both matrix stiffness and adhesiveness on microvascular network (MVN) formation of endothelial cells and the morphology of human mesenchymal stem cells (hMSC). When endothelial cells were encapsulated within 3-D gel matrices without binding sites, little cell elongation and no network formation occurred, regardless of the stiffness. In contrast, matrices containing the RGD binding site facilitated robust MVN formation, and the extent of this MVN formation was inversely proportional to matrix stiffness. Compared with a matrix of the same stiffness with no binding sites, a matrix containing RGD-functionalized peptides resulted in a ∼2.5-fold increase in the average length of network structure, which was used as a quantitative measure of MVN formation. Matrices with hMSC facilitated an increased number and length of cellular projections at higher stiffness when RGD was present, but induced a round morphology at every stiffness when RGD was absent. Taken together, these results demonstrate the ability to control both substrate stiffness and binding site density within 3-D cell-populated gels and reveal an important role for both stiffness and adhesion on cellular behavior that is cell-type specific.

The Q-rich subdomain of the human Ah receptor transactivation domain is required for dioxin-mediated transcriptional activity.

The aryl hydrocarbon receptor (AhR), a basic helix-loop-helix/Per-Arnt-Sim transcription factor, mediates many of the toxic and biological effects of the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin, which include the transcriptional activation of dioxin-responsive genes such as CYP1A1. Many aspects of this process are known; however, the mechanism of transcriptional activation and the proteins that are key to this process remain to be determined. The hAhR has a complex transactivation domain, composed of three potentially distinct subdomains. Deletional analysis of the hAhR transactivation domain indicates that removal of the P/S/T-rich subdomain enhances transcriptional activity, whereas the Q-rich subdomain is critical for hAhR transactivation potential, and the acidic subdomain by itself fails to activate a dioxin response element-driven reporter gene. Deletional analysis of the Q-rich subdomain identified a critical stretch of 23 amino acids between residues 666 and 688 of the hAhR, which are required for transactivation potential. Alanine scanning mutagenesis of this region identified a leucine residue (Leu-678), which is required for hAhR activity. Functional analysis of this point mutant revealed that it is capable of binding ligand, heterodimerization, and subsequent binding to dioxin response elements. Further, when hAhR/L678A and hAhR containing only the acidic subdomain were overexpressed they acted as dominant negative receptors and repressed wild-type hAhR activity. In addition, the hAhR/L678A failed to activate CYP1A1 gene transcription in transfected BP-8 cells and exhibited reduced binding to RIP140 in vitro. Thus, Leu-678 appears to be critical for efficient transactivation activity of the hAhR and appears to disrupt recruitment of co-regulators.

Screening of various Swertia species extracts in primary monolayer cultures of rat hepatocytes against carbon tetrachloride- and paracetamol-induced toxicity.

Swertia chirata Buch-Ham. (Gentianaceae), one of the oldest medicinal herbs of India, is a source of the Indian ayurvedic drug 'chirata' used for the treatment of liver disorders and malarial fevers. In this study, eight species of Swertia were collected. Each of the dry whole plant was extracted into methanol, the aqueous extract of which was sequentially extracted into hexane, chloroform and butanol extracts. The extracts were screened for their anti-hepatotoxic activity against carbon tetrachloride (CCl4) and paracetamol (acetaminophen (AAP)) toxicity in primary monolayer cultures of rat hepatocytes. The primary cultures, 2.5 x 10(6) cells /3 ml medium/60 mm collagen-coated plates, were exposed to 2.5 mM CCl4 or 12 mM AAP in the presence or absence of plant extracts (100 microg/ml culture medium). Cells and medium were harvested after 22 h of treatment for the assay of cellular reduced gluthathione (GSH) content and leakage of lactate dehydrogenase as biological end-points of toxicity. Both CCl4 and AAP at the indicated concentrations reduced GSH by almost 50 and 80%, respectively, while the enzyme leakage was almost 15% above the untreated control. Hexane and methanol extracts of most of the species in general offered relatively good protection. The anti-hepatotoxic activity, nevertheless, was evident in all Swertia species against both the toxicants. However, Swertia purpurascens, Swertia chirata, Swertia paniculata and Swertia cordata exhibited better activity compared with other species investigated. In addition, influence of various extracts (10-100 microg/ml medium) was examined on cellular growth of rat Reuber hepatoma cell line H4IIEC3/G-. Except for the butanol extract of S. chirata, no other extracts exerted toxicity in terms of neutral red uptake by the cells.

Structure-activity relationship of piperine and its synthetic analogues for their inhibitory potentials of rat hepatic microsomal constitutive and inducible cytochrome P450 activities.

Inhibitors of drug metabolism have important implications in pharmaco-toxicology and agriculture. We have reported earlier that piperine, a major alkaloid of black and long peppers inhibits both constitutive and inducible cytochrome P450 (CYP)-dependent drug metabolising enzymes. In the present study, an attempt has been made to prepare several novel synthetic analogues so as to relate various modifications in the parent molecule to the inhibition of CYP activities. Two types of mono-oxygenase reactions arylhydrocarbon hydroxylase (AHH) and 7-methoxycoumarin-O-demethylase (MOCD) have been studied. Inhibition studies were investigated in rat microsomal fraction prepared from untreated, 3MC- and PB- treated rat liver in vitro. Modifications were introduced into the piperine molecule: (i) in the phenyl nucleus, (ii) in the side chain and (iii) in the basic moiety. Thus, 38 compounds have been subjected to such studies, and simultaneously an attempt has also been made to arrive at the structure-activity relationship of synthetic analogues. In general, most of the inhibitory potential of the parent molecule is lost with modification in either of the three components of piperine. Saturation of the side chain resulted in significantly enhanced inhibition of CYP while modifications in the phenyl and basic moieties in few analogues offered maximal selectivity in inhibiting either constitutive or inducible CYP activities. Thus few novel analogues as CYP inactivators have been synthesized which may have important consequences in pharmacokinetics and bioavailability of drugs.

Malnutrition sequela on the drug metabolizing enzymes in male Holtzman rats.

The effect of food restriction on the specific activities of the drug metabolizing enzymes (DME) system was studied in Holtzman male rats by comparing DME activities in 90-day-old control rats fed ad libitum (CO), rats fed 40% restricted food (RF) from the gestation period to the day of sacrifice, and recovered rats (rRF) fed 40% restricted food from period of gestation to 45 days of age and then fed ad libitum until the day of sacrifice. In liver, total cytochrome P450 (CYP) of the RF and rRF groups was higher by approximately 50% and 28%, respectively, than in CO rats. Specific activities of individual CYP monooxygenases (MO) such as CYP2B [7-methoxycoumarin demethylase (MOCD)], CYP1A [aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin deethylase (EORD)], and CYP2E [nitrosodimethylamine demethylase (NDMAd)] were 31, 61, 43, and 56% in RF and 16, 36, 26, and 32% in rRF groups, respectively, more than the CO values. Conjugases such as UDP- glucuronosyltransferases with substrates 3-OH benzo(a)pyrene (UGT1) and 4-hydroxybiphenyl (UGT2) and glutathione S-transferase (GST) with substrate 1-chloro-2,4-dinitrobenzene were higher by 72, 69, and 33% in RF and 28, 38, and 24% in rRF groups, respectively. MO activities (MOCD and EORD) were significantly higher in lung, kidney, and intestine: MOCD by 82, 48, and 45% in RF and 40, 25, and 22% in rRF, respectively; and EORD by 84, 77, and 67% in RF and 40, 33, and 28% in rRF, respectively. However, activity of conjugases (UGT1 and GST) were significantly lower (approximately 35-45%) in RF and rRF rats (approximately 20-30%) than in the CO group in above mentioned extrahepatic tissues. These studies indicate that undernourishment during the period of gestation, weanling, and growth and development of microsomal enzymes produces a sequela of events on the DME in hepatic and extrahepatic tissues that cannot return to the control values even when fed ad libitum.

In vitro assessment of paracetamol-induced toxicity in the rat Reuber hepatoma H4IIEC3/G(-) cell line competent of xenobiotics metabolism.

H4IIEC3/G(-) cells, descendants of rat Reuber hepatoma, were characterized for their response to paracetamol (acetaminophen; AAP) toxicity during log-phase of growth. The cells in culture were found to contain high contents of constitutive and dexamethasone inducible rat cytochrome P4503A besides other CYP members reported earlier. AAP produced dose-dependent decrease in cellular growth (50% at 0.7mm). The drug steadily reduced the activity of UDP-glucuronyltransferase (UGT) towards 3-hydroxybenzo[a]pyrene, decreased the contents of UDP-glucuronic acid (UDPGA) and significantly lowered GSH contents with length of exposure. After 48hr of treatment, the GSH levels registered a fall of 50% while UGT and UDPGA exhibited a moderate decline of less than 25%. Decrease in the conjugation capacity of cells correlated with LDH leakage in the medium. Three compounds of natural origin, silymarin, kutkin and andrographolide at 10-20 mum, offered relatively modest protection ranging from 24 to 55% at best, against the growth inhibitory effect of paracetamol. The hepatoma cells investigated appeared metabolically competent to respond to AAP toxicity and might prove useful for screening of agents against AAP-induced hepatotoxicity.

Effects of silymarin on UDP-glucuronic acid and glucuronidation activity in the rat isolated hepatocytes and liver in relation to D-galactosamine toxicity.

Influence of silymarin on UDP-glucuronic acid (UDPGA) and glucuronidation activity of freshly isolated rat hepatocytes in suspension and in rat liver in vivo was examined. Viability of the hepatocytes (> 85%) was not altered in Hank's balanced salt solution at least for 4 hr at 37 degrees C under oxygen. Silymarin at 0.4 mM depleted UDPGA by more than 60% at the end of 4 hr of incubation, the fall in nucleotide pool was rapid and concentration (0.1-0.4 mM)-dependent. The rate of glucuronidation of 3-OH- benzo(a)pyrene (3-OH-BP) determined simultaneously was also reduced significantly; silybin being 3-times more effective than silymarin. Combination of flavonoids with D-galactosamine (GalN) further attenuated the glucuronidation functions of the cells. The flavonoids also offered strong inhibition of UDP-glucose dehydrogenase (UDP-GDH) activity in the liver cytosolic fraction while the activity in hepatocytes was not affected even after 4 hr of incubation. Interestingly, the GalN- induced strong inhibition of UDP-GDH in isolated hepatocytes was completely abolished by flavonoids. Decrease in UDPGA appeared neither due to the activation of UDPGA-pyrophosphatase activity nor to the inhibition of UDP-GDH activity in hepatocytes. Further, the flavonoids also inhibited hepatic UDP-glucuronyltransferase activity towards 3-OH-BP (UGT) both in vitro and in intact cells. On the contrary, silymarin administered (70 mg/kg body wt; i.p.) to rats for 3 hr increased the hepatic UDPGA by 2-fold while GalN (400 mg/kg body wt) reduced the nucleotide content to 50% of control. Coadministration of silymarin and GalN restored the UDPGA content significantly while the activities of UDP-GDH and UGT were comparable to the untreated control. The results indicated that silymarin elicits differential effects on the rate of glucuronidation and contents of UDPGA in the isolated rat hepatocytes and in liver. The flavonoid counteracted D-GalN-induced lowering of UDPGA presumably by relieving UDP-GDH of in vivo inhibition affected by GalN-metabolite.

Piperine inhibits aflatoxin B1-induced cytotoxicity and genotoxicity in V79 Chinese hamster cells genetically engineered to express rat cytochrome P4502B1.

We have investigated the potential of piperine for inhibiting the activity of cytochrome P4502B1 and protecting against aflatoxin B1 (AFB1) in V79MZr2B1 (r2B1) cells, i.e. V79 Chinese hamster cells engineered for the expression of rat CYP4502B1. The cells were found to contain high activities of 7-methoxycoumarin demethylase (MOCD). Piperine inhibited MOCD in preparations of r2B1 cells with an IC50 of approximately 10 microM. The cells in culture dealkylated 7-methoxycoumarin (MOC) to 7-OH-coumarin linearly, at least for 12 h, where piperine produced concentration-dependent inhibition with IC50 < 30 microM. The time required for maximal inhibition was approximately 8 h with both 30 and 60 microM concentrations of piperine used. AFB1 at 0.1-20 microM caused a concentration dependent decrease in the amount of DNA and an increase in the formation of micronuclei (MN). The mycotoxin at 10 microM reduced DNA by approximately 30% and increased MN appearance by 20-fold against the background level of 7 MN per 500 nuclei. Piperine at 60 microM completely counteracted cytotoxicity and formation of MN by 10 microM AFB1 and reduced the toxic effects of 20 microM AFB1 by > 50%. The results suggest that: (i) Piperine is a potent inhibitor of rat CYP4502B1 activity; (ii) AFB1 is activated by r2B1 cells to cytotoxic and genotoxic metabolites; and (iii) piperine counteracts CYP4502B1 mediated toxicity of AFB1 in the cells and might, therefore, offer a potent chemopreventive effect against procarcinogens activated by CYP4502B1.

Piperine impairs cytochrome P4501A1 activity by direct interaction with the enzyme and not by down regulation of CYP1A1 gene expression in the rat hepatoma 5L cell line.

Modulation of benzo(a)pyrene (BP) metabolism and regulation of CYP1A1 gene expression by piperine in 5L cells in culture was studied. Treatment of cultures with 60 microM piperine for different time periods inhibited metabolism of BP by 50% within 4-8 h. Piperine uptake in 5L cells attained saturation plateau at 8 h and this related with the maximal impairment of BP metabolism. Exposure of cell cultures to piperine for 24 h indicated activation of CYP1A1 gene transcription. There was a 10-fold increase in CYP1A1mRNA and an approximately 7-fold increase in arylhydrocarbon hydroxylase (AHH) activity, while treatment with benzanthacene (BA) increased CYP1A1mRNA by 86- and AHH by 56-fold. Combined treatment with BA plus piperine further increased CYP1A1mRNA contents by about 25%. The BA-inducible AHH activity, however, registered a decrease of about 45%. Piperine neither destroyed CYP1A1-protein nor affected the total cellular protein contents. Exposure of cultures to 0.01 to 3.0 microM trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene for 24 h reduced maximum cellular growth by about 50%. Piperine at 60 microM offered full protection against the diol cytotoxicity. The results, suggest that piperine mediated inhibition of the AHH activity and consequent suppression of the procarcinogen activation is the result of direct interaction of piperine with CYP1A1-protein and not because of down regulation of the CYP1A1 gene expression.

Piperine, a major ingredient of black and long peppers, protects against AFB1-induced cytotoxicity and micronuclei formation in H4IIEC3 rat hepatoma cells.

We studied the effect of piperine on the cytotoxicity and genotoxicity of aflatoxin B1 (AFB1) in rat hepatoma cells H4IIEC3/G-(H4IIE) using cellular growth and formation of micronuclei as endpoints. Piperine was earlier shown to inhibit cytochrome P-450-dependent aryl hydrocarbon hydroxylase and 7-methoxycoumarin demethylase activities in preparations of these cells with 1/2 maximum inhibition at 30-50 microM (Singh J. and Reen R.K., Current Science, 66, 365-369, 1994). The results of the present study showed that AFB1 inhibited the growth of H4IIE cells with an ED50 of 15 nM. Piperine markedly reduced the toxicity of the mycotoxin. Thus at 100 microM piperine largely restored the rate of growth of the cells. Likewise, piperine reduced the AFB1-induced formation of micronuclei in a concentration-dependent manner. Piperine itself was not toxic to the cells up to a concentration of almost 100 microM. The results suggest, that piperine is capable of counteracting AFB1 toxicity by suppressing cytochromes P-450 mediated bioactivation of the mycotoxin.

Impairment of UDP-glucose dehydrogenase and glucuronidation activities in liver and small intestine of rat and guinea pig in vitro by piperine.

The effects of piperine, a major ingredient of black pepper, on UDP-glucose dehydrogenase (UDP-GDH) and glucuronidation potentials of rat and guinea pig liver and intestine were studied. Piperine caused a concentration-related strong inhibition of UDP-GDH (50% at 10 microM) reversibly and equipotently, in both tissues. Partially purified rat liver UDP-GDH was used to obtain the kinetic values at pH optima of 9.4 and 8.6. At pH 9.4: KmUDP-glucose = 15 microM, Vmax = 5.2 nmol NADH/min/mg protein, Ki = 6 microM. With NAD, a Ki of 16 microM was obtained. At pH 8.6: Km = 35 microM, Vmax = 7.5 nmol, Ki = 15 microM. In all of these cases, piperine caused non-competitive inhibition. Data from structure-activity comparisons of piperine analogs indicated that the presence of conjugated double bonds in the side chain of the molecule is a factor in piperine inhibition. However, the UDP-glucuronic acid (UDPGA) contents were decreased less effectively by piperine in isolated rat hepatocytes compared with enterocytes of guinea pig small intestine. Piperine at 50 microM caused a marginal decrease of UDPGA in hepatocytes when the rate of glucuronidation of 3-hydroxybenzo[a]pyrene (3-OH-BP) decreased by about 40%. The decrease obtained at 10 microM piperine in intestinal cells was comparable to that obtained at 50-100 microM in hepatocytes. UDP-glucuronosyltransferase (UGT) activities towards 3-OH-BP (UGT1A1) and 4-OH-biphenyl (UGT2B1) were also determined. Piperine did not affect the rate of glucuronidation of 4-OH-biphenyl in rat liver, whereas that of 3-OH-BP was impaired significantly. In guinea pig small intestine, both these activities were inhibited significantly requiring less than 25 microM piperine to produce a more than 50% inhibition of UGT(s). The results suggested that (i) piperine is a potent inhibitor of UDP-GDH, (ii) inhibition is offered exclusively by the conjugated double bonds of the molecule, and (iii) piperine exerts stronger effects on intestinal glucuronidation than in rat liver.

In vitro and in vivo inhibition of pulmonary cytochrome P450 activities by piperine, a major ingredient of piper species.

In vitro and in vivo modulation of drug metabolizing enzymes by piperine was investigated in microsomes of rats and guinea pigs. In vitro piperine caused concentration related inhibition (50% at 100 microM) of arylhydrocarbon hydroxylase (AHH) and 7-ethoxycourmarin deethylase (7ECDE) activities, which were comparable in control and 3-methylcholanthrene (3MC) treated rats. In guinea pig microsomes however, piperine caused strong inhibition at lower concentrations (35% at 10 microM) and relatively much lesser inhibition with further increase in piperine concentrations. A Dixon plot of the kinetic data of both AHH and 7ECDE indicated noncompetitive inhibition with a Ki of approx. 100 microM. In vivo, piperine given at a dose of 25 mg/kg body wt to rats caused a maximal inhibition at 1 hr of both the enzymes, while only AHH returned to normal value within 4 hr. Similarly, upon daily treatment of piperine (15 mg/kg body wt) to rats for 7 days, 7ECDE was consistently inhibited, while AHH showed faster recovery. Piperine thus appeared to cause differential inhibition of two forms of cytochrome P450 and thus would accordingly affect the steady-state level of those drugs metabolized by these pulmonary forms of cytochromes P450.

Dealkylation of 7-methoxycoumarin as assay for measuring constitutive and phenobarbital-inducible cytochrome P450s.

Six alkyl ethers of 7-hydroxycoumarin, ranging from methoxy- to hexoxycoumarin, were studied for their NADPH-dependent metabolism by liver microsomes of male rats treated with phenobarbital (PB) or 3-methyl-cholanthrene (MC). The six alkyl ethers were metabolized by both types of microsomes, forming 7-hydroxycoumarin as the major product. Among the test compounds, 7-methoxycoumarin was unusual in that its dealkylation was inducible only by PB and not by MC. PB increased 7-methoxycoumarin-O-demethylase (MOCD) activity about four- to eightfold. Metyrapone strongly inhibited MOCD in PB-treated microsomes but not in MC-treated microsomes. Similarly, monoclonal antibodies directed toward PB-induced cytochrome P450s selectively suppressed MOCD in PB-treated microsomes. MOCD activity was observed in preparations of SD1 cells containing only cytochrome P450IIB1, while it was not found in preparations of XEM1 cells containing only cytochrome P450IA1. Demethylation of 7-methoxycoumarin was also mediated by the constitutive cytochrome P450 form(s) of liver, lung, small intestine, and kidney (in decreasing order). PB increased MOCD activity of small intestine by 40% but was without effect on the dealkylation activity of lung and kidney. MOCD activity was also detectable in differentiated rat hepatoma lines H4IIEC3 and 2sFou. The studies indicate that dealkylation of 7-methoxycoumarin is a highly sensitive, simple, and practical assay for estimating constitutive and PB-inducible cytochrome P450-dependent monooxygenase activities.