Efficacy and Safety of FX201, a Novel Intra-Articular IL-1Ra Gene Therapy for Osteoarthritis Treatment, in a Rat Model.

Osteoarthritis (OA) is a disabling, degenerative disease characterized by progressive cartilage and bone damage. There remains a need for local therapies that, following a single injection, can provide long-term pain relief and functional improvement and potentially delay disease progression. FX201 is a novel, intra-articular (IA), interleukin-1 receptor antagonist (IL-1Ra) gene therapy in development for the treatment of OA. In this study, we assessed the efficacy, biodistribution, and safety of helper-dependent adenovirus (HDAd)-ratIL-1Ra, the rat surrogate of FX201, and the biodistribution of FX201, in the anterior cruciate ligament transection (ACLT) rat OA model. A single IA injection of HDAd-ratIL-1Ra administered 7 days post-ACLT mitigated OA-related changes to cartilage, bone, and the synovial membrane at week 12 following surgery. Furthermore, FX201 and HDAd-ratIL-1Ra persisted for at least 92 days in the injected joint and proximal tissues with minimal evidence of vector spreading peripherally. Finally, HDAd-ratIL-1Ra showed a favorable safety profile without any local or systemic adverse effects. In conclusion, HDAd-ratIL-1Ra demonstrated local therapeutic and disease-modifying effects and was well tolerated, supporting further clinical development of FX201.

Preclinical and Clinical Characteristics of the Trichuricidal Drug Oxantel Pamoate and Clinical Development Plans: A Review.

Soil-transmitted helminths (Ascaris lumbricoides, hookworm and Trichuris trichiura) infect about one-fifth of the world's population. The currently available drugs are all highly efficacious against A. lumbricoides. However, they are only moderately efficacious against hookworm and poorly efficacious against T. trichiura. Oxantel, a tetrahydropyrimidine derivative discovered in the 1970s, has recently been brought back to our attention given its high efficacy against T. trichiura infections (estimated 76% cure rate and 85% egg reduction rate at a 20 mg/kg dose). This review summarizes the current knowledge on oxantel pamoate and its use against T. trichiura infections in humans. Oxantel pamoate acts locally in the human gastrointestinal tract and binds to the parasite's nicotinic acetylcholine receptor (nAChR), leading to a spastic paralysis of the worm and subsequent expulsion. The drug is metabolically stable, shows low permeability and low systemic bioavailability after oral use. Oxantel pamoate was found to be safe in humans, with only a few mild adverse events reported. Several clinical trials have investigated the efficacy of this drug against T. trichiura and suggest that oxantel pamoate is more efficacious against T. trichiura than the currently recommended drugs, which makes it a strong asset to the depleted drug armamentarium and could help delay or even prevent the development of resistance to existing drugs. We highlight existing data to support the use of oxantel pamoate against T. trichiura infections.

Toxicological testing of allogeneic secretome derived from peripheral mononuclear cells (APOSEC): a novel cell-free therapeutic agent in skin disease.

A cell-free approach using secretomes derived from stem cells or peripheral blood mononuclear cells is an active area of regenerative medicine that holds promise for therapies. Regulatory authorities classify these secretomes as biological medicinal products, and non- clinical safety assessment thus falls under the scope of ICH S6. A secretome of stressed peripheral blood mononuclear cells (APOSEC) was successfully tested in a toxicology program, supporting clinical use of the new drug candidate. Here, to allow for topical, dermal treatment of patients with diabetic foot ulcer, several non-clinical safety studies were performed. Acute toxicity (single dose) and neuropharmacological screening were tested intravenously in a rat model. Risk for skin sensitisation was tested in mice. A 4-week intravenous toxicity study in mice and a 4-week subcutaneous toxicity study in minipigs were conducted to cover the clinical setting and application in a rodent and a non-rodent model. Acute and repeated-dose toxicity studies show that APOSEC administered intravenously and subcutaneously does not involve major toxicities or signs of local intolerance at levels above the intended total human maximal dose of 3.3 U/kg/treatment, 200 U/wound/treatment, and 100 U/cm2/treatment. The non-clinical data support the safe topical use of APOSEC in skin diseases related to deficient wound healing.

Pharmacology, toxicology and clinical safety of glycopyrrolate.

The clinical use of the anticholinergic glycopyrrolate dates back to the early 1960s when it was first approved in the U.S. Since then, oral and inhalation formulations have been developed as therapeutic agents inhibiting the muscarinic acetylcholine receptor in various indications including chronic obstructive pulmonary disease (COPD), excessive salivation, and peptic ulcers. More recently, topical formulations of glycopyrrolate (GPB, also known as glycopyrronium bromide) have gained interest as a treatment option for excessive sweating (hyperhidrosis). The U.S. Food and Drug Administration (FDA) approved the first topical glycopyrronium product for the treatment of hyperhidrosis in 2018. Glycopyrrolate, as a quaternary amine, shows minimal penetration of the blood brain barrier which limits CNS side effects. In addition, lack of phototoxicity, genotoxicity and carcinogenicity makes it suitable for chronic indications. The information on the nonclinical and clinical safety profile of glycopyrronium supporting various therapeutically approved uses has been obtained from published literature, our own data as well as summary documents issued by regulatory bodies. Collectively, these data support the conclusion that the benefits of glycopyrronium generally outweigh the risks in chronic use indications that require muscarinic receptor antagonism to provide therapeutic effects.

Non-clinical safety evaluation of intranasal iota-carrageenan.

Carrageenan has been widely used as food additive for decades and therefore, an extended oral data set is available in the public domain. Less data are available for other routes of administration, especially intranasal administration. The current publication describes the non-clinical safety and toxicity of native (non-degraded) iota-carrageenan when applied intranasally or via inhalation. Intranasally applied iota-carrageenan is a topically applied, locally acting compound with no need of systemic bioavailability for the drug's action. Animal experiments included repeated dose local tolerance and toxicity studies with intranasally applied 0.12% iota-carrageenan for 7 or 28 days in New Zealand White rabbits and nebulized 0.12% iota-carrageenan administered to F344 rats for 7 days. Permeation studies revealed no penetration of iota-carrageenan across nasal mucosa, demonstrating that iota-carrageenan does not reach the blood stream. Consistent with this, no relevant toxic or secondary pharmacological effects due to systemic exposure were observed in the rabbit or rat repeated dose toxicity studies. Data do not provide any evidence for local intolerance or toxicity, when carrageenan is applied intranasally or by inhalation. No signs for immunogenicity or immunotoxicity have been observed in the in vivo studies. This is substantiated by in vitro assays showing no stimulation of a panel of pro-inflammatory cytokines by iota-carrageenan. In conclusion, 0.12% iota-carrageenan is safe for clinical use via intranasal application.

Cadmium stress stimulates tissue turnover in Helix pomatia: increasing cell proliferation from metal tolerance to exhaustion in molluscan midgut gland.

In terrestrial pulmonate snails, cadmium (Cd) uptake leads to the induction of a Cd-specific metallothionein isoform (Cd-MT) that protects against adverse interactions of this toxic metal ion. Increasing concentrations of Cd cause increased individual mortality possibly linked to pathological alterations in the snail midgut gland. Histological, immuno-histochemical, and electron-microscopic methods in combination with tissue metal analyses and quantification of MT induction parameters were applied to the midgut gland of Cd-exposed Roman snails (Helix pomatia). Conspicuous concentration-dependent alterations occurred in this organ, including the metal-induced increase of Cd-MT concentration and manifestation of Cd-MT mRNA precipitations in all midgut gland cell types. The most evident alteration was an increase of cellular turnover reflected by enhanced cell proliferation. Intensified vesiculation of endoplasmic reticulum was noted in basophilic cells and an increasing formation of lipofuscin granules in excretory cells. At the highest Cd concentrations, mitochondrial membranes were disrupted in basophilic cells, and lipofuscin granules were released from excretory cells into the midgut gland tubular system. Some of these alterations (e.g., increased cell proliferation rate, vesiculation of endoplasmic reticulum) detected at low Cd concentrations were interpreted as adaptive response processes enhancing the tolerance of exposed individuals to metal stress. Cellular alterations at higher Cd concentrations (e.g., mitochondrial structural damage) clearly represented ongoing irreversible cellular disruption. Combined evaluation of cellular biomarkers and MT saturation levels indicated that the transition from stress resistance to depletion of resistance capacity occurred above a threshold of 0.8 micromol Cd/g dry weight in the midgut gland of H. pomatia. At these Cd concentrations, Cd-MT was saturated with Cd(2+) ions, whereas at the cellular level, structural alterations turned into pathological deterioration.

Pre-clinical safety evaluation of pyrrolidine dithiocarbamate.

Pyrrolidine dithiocarbamate (PDTC) was examined for its potential in the intranasal treatment of human rhinovirus infections. Prior to clinical testing, a comprehensive non-clinical programme was performed to evaluate the general toxicity of PDTC. The animal experiments included investigations in rodents with study durations ranging from single dose to repeated dosing over a period of 28 days. The routes of administration were intranasal, inhalative, oral and intravenous for single-dose toxicity and pharmacokinetic studies, and intranasal for repeated dose studies. Blood and tissue samples were obtained from PDTC-treated rats to analyse pharmacokinetics and tissue distribution. Accumulation of selected metals due to PDTC treatment was examined in liver, brain, nerves and fat tissues.

Non-clinical development of cancer vaccines: regulatory considerations.

This paper discusses regulatory requirements essential during the non-clinical development of cancer vaccines. DNA vaccines and vaccines containing monoclonal antibodies are specifically addressed. ICH, CHMP, FDA, and WHO guidance documents in addition to scientific literature are reviewed and the regulatory framework, including respective EMEA and the FDA divisions responsible for review and assessment of cancer vaccines, is described. Selection criteria for an appropriate animal model for efficacy and/or toxicity studies are discussed.

Copper in Helix pomatia (Gastropoda) is regulated by one single cell type: differently responsive metal pools in rhogocytes.

Like all other animal species, terrestrial pulmonate snails require Cu as an essential trace element. On the other hand, elevated amounts of Cu can exert toxic effects on snails. The homeostatic regulation of Cu must therefore be a pivotal goal of terrestrial pulmonates to survive. Upon administration of Cu, snails accumulate the metal nearly equally in most of their organs. Quantitative studies in connection with HPLC and electrospray ionization mass spectrometry reveal that a certain fraction of Cu in snails is bound to a Cu-metallothionein (Cu-MT) isoform that occurs in most organs at constant concentrations, irrespective of whether the animals had been exposed to physiological or elevated amounts of Cu. In situ hybridization demonstrates that at the cellular level, the Cu-binding MT isoform is exclusively expressed in the so-called pore cells (or rhogocytes), which can be found in all major snail organs. The number of pore cells with Cu-MT mRNA reaction products remains unaffected by Cu exposure. Rhogocytes also are major storage sites of Cu in a granular form, the metal quickly entering the snail tissues upon elevated exposure. The number of rhogocytes with granular Cu precipitations strongly increases upon Cu administration via food. Thus, whereas Cu-MT in the rhogocytes represents a stable pool of Cu that apparently serves physiological tasks, the granular Cu precipitations form a second, quickly inducible, and more easily available pool of the metal that serves Cu regulation by responding to superphysiological metal exposure.

Apoptosis in stages of mouse hepatocarcinogenesis: failure to counterbalance cell proliferation and to account for strain differences in tumor susceptibility.

C3H/He and B6C3F1 show much higher liver cancer susceptibility than C57BL/6J mice. We studied the hypothesis that this difference might result from failure of apoptosis. Hepatocarcinogenesis was induced by a single dose of N-nitrosodiethylamine (NDEA), followed by phenobarbital (PB) for up to 90 weeks. We observed (1) earlier appearance of putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and carcinoma (HCC) in C3H/He than in C57Bl/6J mice and (2) an increase of hepatocellular DNA synthesis in C3H/He and C57Bl/6J mice, compared to normal liver, via PPF and HCA to HCC. PB enhanced DNA synthesis and growth of PPF, in the C3H/He strain only, and of HCA and HCC of both strains. Apoptoses were rare in unaltered livers as well as in preneoplastic lesions, but tended to increase in HCA and HCC of both strains. PB lowered apoptotic activity in PPF of C3H/He mice, but enhanced it in HCA and HCC of C57Bl/6J mice at late stages. In conclusion, the strain difference in growth rates of PPF and tumors is largely determined by higher rates of cell proliferation in C3H/He mice, with and without promotion by PB. Moreover, in C57Bl/6J mice the promoting effect of PB was restricted to HCA and HCC and was not seen in PPF. Apoptosis was generally low and was not a major cause of the strain difference in tumor susceptibility. In contrast with rat liver, inhibition of apoptosis appears to be a minor determinant of tumor promotion in mice.

Mechanisms of cadmium toxicity in terrestrial pulmonates: programmed cell death and metallothionein overload.

A sublethal dose of cadmium (Cd2+) administered via the diet during short-term exposure over 10 d induced programmed cell death in the hepatopancreas of the terrestrial pulmonate snail Helix pomatia. Condensed cell residues were predominantly phagocytosed by calcium cells, suggesting a specific function of these epithelial cells in metal detoxification or in clearing the organ of cellular debris from cell death. The considerable cell loss recorded by histological analysis was accompanied by enhanced cell proliferation. Intoxication with Cd was further associated with the pronounced abundance of residual bodies, predominantly recorded in excretory cells, and with pathological changes in the endoplasmic reticulum. During long-term Cd exposure, mortality increased with increasing Cd concentrations in the diet, as demonstrated by feeding experiments in the laboratory. Lethal effects of Cd appeared to be correlated with Cd overloading of the Cd-specific metallothionein isoform (Cd-MT), isolated and characterized previously from the animal's hepatopancreas. Stoichiometric analysis shows that the capacity of Cd-MT to bind six molar equivalents of Cd corresponds to a tissue Cd concentration of approximately 4 micromol/g dry weight. At this tissue concentration, all high-affinity metal-binding sites of Cd-MT are occupied by Cd2+. Cadmium exposure beyond this level gives rise to progressive destabilization of Cd-MT cluster structure in vitro, resulting in increasing proportions of weakly bound, or even unbound, Cd2+ ions. Our results suggest that in vivo, the observed overburdening of Cd-MT with Cd2+ reduces the viability of affected animals.

Isoform-specific quantification of metallothionein in the terrestrial gastropod Helix pomatia. II. A differential biomarker approach under laboratory and field conditions.

The two function-specific metallothionein (MT) isoforms characterized from the midgut gland and mantle tissue of Helix pomatia differ substantially in their metal-binding preferences, as well as molecular and biochemical features. These differences make them potential candidates for biomarker studies based on a differential, isoform-specific approach. To prove this hypothesis, induction experiments with two metals (Cd and Cu) that are normally bound by the two isoforms were compared with a range of organic chemicals and physical stressors under laboratory conditions to test the responsiveness of the two isoforms to the stressors applied. In addition, field studies were conducted with Roman snails and substrate samples collected from different metal-contaminated sites in Austria to test the suitability of the two isoforms as biomarkers under field conditions. The results of these combined laboratory and field studies confirmed the validity of the biomarker approach with the two metal- and tissue-specific isoforms. It is demonstrated that the Cd-binding MT specifically and exclusively responds to Cd exposure by increasing concentrations, whereas the Cu-binding MT isoform decreases in its concentration upon exposure to physical stress (X-ray irradiation and cold). This suggests researchers should adopt, under certain preconditions, a dual biomarker approach by combining the simultaneous quantification of Cd-MT concentrations in the midgut gland as a biomarker for Cd pollution and of Cu-MT concentration in the mantle as a biomarker for the impairment of snails by additional physical stressors.

Isoform-specific quantification of metallothionein in the terrestrial gastropod Helix pomatia. I. Molecular, biochemical, and methodical background.

The biomarker concept is an important approach of modern ecotoxicology based on the detection and quantification of molecular, biochemical, cellular, or physiological alterations in organisms as the result of exposure to toxic compounds. In the present study, the biomarker potential of two metallothionein (MT) isoforms from hepatopancreas and mantle tissue of the Roman snail, Helix pomatia L., is evaluated. This species possesses two MT isoforms with specific functions: The Cd-specific isoform (Cd-MT) is predominantly induced and expressed in the hepatopancreas upon Cd exposure. It binds and inactivates this potentially toxic trace element. The second isoform is Cu-specific (Cu-MT), primarily expressed in the mantle tissue and responsible for the homeostatic regulation of Cu. The differential expression and metal-specificity of the two isoforms make them potential candidates for a dual MT biomarker system based on an isoform-specific approach. This study shows that Cd-MT and Cu-MT of H. pomatia can reliably be quantified by a differential metal saturation approach that could be used for environmental biomarker studies. The approach works by virtue of the following preconditions: The two isoforms differ from each other with respect to their molecular and biochemical features, they show metal-specific binding preferences, they are organ-specific, they exhibit distinct induction patterns in response to the metals to which they bind, and their biological and functional background is thoroughly known.

Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia).

The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined.

Cd accumulation and Cd-metallothionein as a biomarker in Cepaea hortensis (Helicidae, Pulmonata) from laboratory exposure and metal-polluted habitats.

Cepaea hortensis is a widespread terrestrial pulmonate, contributing significantly to element fluxes in soil ecosystems. Due to its capacity of accumulating certain trace elements in its tissues, Cepaea hortensis can serve as a biological indicator of metal accumulation in contaminated areas. In response to Cd exposure this species and related helicid pulmonates are also able to synthesize an inducible, Cd-binding metallothionein (MT) isoform specifically serving in binding and detoxification of this metal. As shown by field-collected garden-snails from a metal-contaminated site near a zinc smelting works in Avonmouth (UK) and an unpolluted reference site in Reutte (Tyrol, Austria), Cd and Cd MT concentrations in midgut gland of C. hortensis from these sites increased with rising Cd concentrations in the soil substrate from the same contaminated sites. By combining the results of these field data with laboratory experiments it appears that midgut gland Cd-MT of Cepaea hortensis seems to fulfil the criteria of a successful biomarker in many respects. First, the synthesis of the protein can rapidly be induced by Cd exposure. Second, the level of Cd MT induction in C. hortensis directly reflects the intensity of metal exposure. Third, the induced signal of increased Cd-MT concentration in C. hortensis is persistent over extended periods of time. Fourth, the Cd-MT signal in C. hortensis seems to be very specific for Cd exposure. Regression analyses demonstrate that tissue levels of Cd and Cd MT in C. hortensis depend on Cd concentrations in the substrate which is represented by either soil or plant material on which snails normally feed. In both cases the best fit for this dependence is exhibited by a semi-logarithmic relationship, with substrate (soil or plant feed) concentrations expressed on a logarithmic scale. It is concluded that C. hortensis and other related pulmonates can successfully be used either as biological indicators of Cd accumulation, or as key species in biomonitoring studies focusing on Cd-MT induction as a biomarker for Cd exposure.

Induction of apoptosis in mouse liver adenoma and carcinoma in vivo by transforming growth factor-beta1.

PURPOSE: In the liver, transforming growth factor beta-1 (TGF-beta1) constitutes a major negative growth regulating factor involved in the control of cell numbers; failure of this control mechanism has been associated with the development of liver cancer. Since no reports on the in vivo effects of exogenously administered TGF-beta1 on apoptosis in liver tumors have been published yet, we studied hepatocyte sensitivity to the proapoptotic action of TGF-beta1 in stages of chemically induced mouse liver carcinogenesis. METHODS: Mouse liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w., i.p.) to 5-week-old B6C3F1 mice. After 2 weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w) for 85 weeks. Four hours before being killed mice received a single dose of TGF-beta1 (56 microg or 200 microg TGF-beta1/kg of b.w., injected into the tail vein). Quantitative histological analysis of mitosis and apoptosis in normal liver tissue (NL), putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) was performed on H&E-stained liver sections. RESULTS: In NDEA and NDEA + PB-treated mice, NL exhibited a very low incidence of apoptosis and mitosis, which increased in HCA and HCC. In the lesions apoptoses ranged between 0.03 and 0.6%. Two hundred micrograms of TGF-beta1/kg stimulated apoptoses in NL as well as in neoplastic lesions (significant increase in NL, HCA, and HCC); the most pronounced proapoptotic action of TGF-ss1 was observed in lesions of NDEA+PB pretreated mice (about 1.7%). Fifty-six microg TGF-beta1/kg had no detectable effect on apoptosis. CONCLUSION: These observations indicate that during chemically induced liver carcinogenesis in B6C3F1 mice basal rates of apoptoses in adenoma and carcinoma are higher than in normal liver and can be further increased by a proapoptotic cytokine.

Overexpression of activin beta(C) or activin beta(E) in the mouse liver inhibits regenerative deoxyribonucleic acid synthesis of hepatic cells.

Activins are dimeric growth factors composed of beta-subunits, four of which have been isolated so far. Whereas activin beta(A) and beta(B) are expressed in many tissues, the expression of activin beta(C) and beta(E) is confined to the liver. To date no biological role or activity has been assigned to activins formed from beta(C) or beta(E) subunits (activin C and E). Because activin A (beta(A)beta(A)), among its various functions in other tissues, appears to be a negative regulator of liver growth, we hypothesized a similar role for activin C and E. Using a nonviral gene transfer system we specifically delivered genes encoding activin beta(C), beta(E), or beta(A) to the mouse liver. The mRNA analysis and reporter gene coexpression both indicated a reproducible temporal and spatial transgene expression pattern. The effects of activin overexpression were studied in the context of a regenerative proliferation of hepatic cells, a result of the tissue damage associated with the hydrodynamics based gene transfer procedure. Activin beta(C), beta(E), or beta(A) expression, all temporarily inhibited regenerative DNA synthesis of hepatocytes and nonparenchymal cells, though to a varying degree. This first report of a biological activity of activin C and E supports an involvement in liver tissue homeostasis and further emphasizes the role of the growing activin family in liver physiology.

Localization and quantification of Cd- and Cu-specific metallothionein isoform mRNA in cells and organs of the terrestrial gastropod Helix pomatia.

A quantitative assay based on real-time detection polymerase chain reaction (rtdPCR) was applied to analyze basal and metal-induced mRNA levels of two metallothionein (MT) isoforms (Cd-MT and Cu-MT) in organs of the terrestrial gastropod Helix pomatia. The results show that specific Cd-MT mRNA levels increase with Cd tissue burden, identifying hepatopancreas and gut as the main organs of Cd accumulation and, accordingly, the predominant organs of Cd-MT mRNA expression. In situ hybridization localized this isoform in epithelial cells of hepatopancreas, gut, and kidney. In contrast to the observed Cd-dependent inducibility of the Cd-binding MT isoform, gene expression of the Cu-binding MT could not be induced by either Cd or Cu exposure. Only very low mRNA amounts of the Cu-MT isoform were found in snail hepatopancreas and kidney, whereas the mantle exhibited high basal mRNA levels of this isoform. In situ localization revealed that the Cu-MT gene expression was restricted to one cell type, the so-called rhogocytes, which are present to various extents in the different organs examined. These results suggest a metal-specific sharing of functions between the two MT isoforms. The Cd-MT isoform apparently plays a crucial role in Cd detoxification, as demonstrated by the inducibility of this isoform, as well as its specific localization in the main metabolic and Cd storing organs. The predominant presence of Cu-MT in rhogocytes of snail mantle strengthens the hypothesis that this isoform may regulate Cu availability in hemocyanin synthesis.

Chemoprevention of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced colonic and hepatic preneoplastic lesions in the F344 rat by cruciferous vegetables administered simultaneously with the carcinogen.

The aim of this study was to investigate the chemopreventive effects of widely consumed cruciferous vegetables, namely Brussels sprouts and red cabbage towards 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced preneoplastic lesions [liver glutathione-S-transferase placental positive (GST-P(+)) foci and colonic aberrant crypt foci (ACF)]. Male F344 rats were treated with IQ (100 mg/kg bw/g) on 10 alternating days and received drinking water supplemented with Brussels sprouts and red cabbage juices (5% v/v) before and during the carcinogen treatment. From each vegetable two different cultivars were tested. Brussels sprouts reduced the frequency of IQ-induced aberrant foci in both organs (41-52% in the colon and 27-67% in the liver). Also, Brussels sprouts drastically diminished (85-91%) the size of liver GST-P(+) foci, but no such effect was seen in the colon. With red cabbage, the size of liver GST-P(+) foci was markedly reduced (41-83%) whereas the foci frequency was only moderately decreased (19-50%). No protection was seen in the colon after treatment with red cabbage. Cooking (10 min, 100 degrees C) of the vegetables had no influence on their protective effects. The stronger chemoprotective effects of Brussels sprouts may be due to the fact that the overall glucosinolate contents were substantially (2-3-fold) higher than those of the cabbage cultivars, but it was not possible to attribute the reduction of preneoplastic lesions to specific glucosinolates. The activities of hepatic UDP-glucuronosyltransferase form 2 (UDPGT-2) and cytochrome P4501A2 were increased by both vegetables. The induction effect of Brussels sprouts on the activity of UDPGT-2 was more marked than that of the red cabbage cultivars, suggesting that increased glucuronidation of IQ may account for the reduction of the preneoplastic lesions. Our findings support the assumption that Brassica vegetables protect against the carcinogenic effects of heterocyclic amines.

Cellular gene dose and kinetics of gene expression in mouse livers transfected by high-volume tail-vein injection of naked DNA.

Direct gene transfer to mammalian tissues has significant potential for biomedical research and gene therapy. Recently, the efficient transfer of naked plasmid DNA to the mouse liver by a rapid high-volume tail-vein injection was reported. We carried out a systematic analysis of the dose and time dependence of the expression of the Escherichia coli beta-galactosidase gene transferred by this technique. Surprisingly, the DNA concentration of the administered solution determined primarily the cellular gene dose and, hence, the expression of the transgene in individual hepatocytes, while the number of transfected cells was largely independent of the supplied plasmid mass. Transgene expression was transient: after a rapid onset and a peak at 8 h past injection, it gradually declined and was no longer detectable 4 weeks later. Although gene transfer was accompanied by tissue damage and subsequent regenerative proliferation, the decline in transgene expression was not due to increased hepatocyte turnover or to promoter downregulation, but instead cells apparently lost the plasmid DNA. Furthermore, we show that "nakedness" of the injected DNA is indeed a prerequisite for efficient transfer by the hydrodynamics-based procedure. Our data provide important clues for the successful use of this gene transfer technique, and may point directions for studies on the underlying mechanisms.