Susanna Penco

Changes of CYP1A1, GST, and ALDH3 enzymes in hepatoma cell lines undergoing enhanced lipid peroxidation

Hepatoma cells show alterations in the response to oxidative stress (decreased lipid peroxidation) and in xenobiotic metabolism enzymes (decreased P450, increased GST and ALDH3). This study examined the effect of lipid peroxidation on the expression of the above enzymes in two rat hepatoma cell lines (MH(1)C(1) and 7777). To induce oxidative stress, cells were exposed to arachidonic acid (to increase lipid peroxidation substrate) and/or to beta-naphthoflavone (to increase CYP450), and treated with one dose of iron/histidine. The cells, that were still viable after the challenge, were refed with the culture medium and CYP1A1, GST, and ALDH3 enzymes monitored for 1, 6, 12, and 24 h. Treatments that increased markers indicative of lipid peroxidation are associated with a decrease in enzyme activities, which was permanent for CYP1A1 and transient for the other enzymes. We speculate from these data that aldehydic byproducts of lipid peroxidation may be responsible for these effects. Thus, restoration of lipid peroxidation in hepatoma cells seems to induce a rapid adaptation to oxidative stress, which is achieved by a simultaneous decrease of reactive oxygen species production and an increase in the two main enzymes involved in the removal of the aldehydic products of lipid peroxidation.

Mechanisms for reduced pulmonary diffusing capacity in haematopoietic stem-cell transplantation recipients

Lung diffusing capacity for CO (DLCO) is compromised in haematopoietic stem-cell transplantation (HSCT) recipients. We derived alveolar-capillary membrane conductance (DM,CO) and pulmonary capillary volume (VC) from DLCO and diffusing capacity for NO (DLNO). Forty patients were studied before and 6 weeks after HSCT. Before HSCT, DLNO and DLCO were significantly lower than in 30 healthy controls. DM,CO was ∼40% lower in patients than in controls (p<0.001), whereas VC did not differ significantly. After HSCT, DLNO and DM,CO further decreased, the latter by ∼22% from before HSCT (p<0.01) while VC did not change significantly. Lung density, serum CRP and reactive oxygen metabolites were significantly increased, with the latter being correlated (R2=0.71, p<0.001) with the decrement in DLNO. We conclude that DLNO and, to a lesser extent, DLCO are compromised before HSCT mainly due to a DM,CO reduction. A further reduction of DM,CO without VC loss occurs after HSCT, possibly related to development of oedema, or interstitial fibrosis, or both.

Comparative evaluation of cytotoxicity and metabolism of four aldehydes in two hepatoma cell lines

The metabolism of acetaldehyde (ACA), benzaldehyde (BA), propionaldehyde (PA) and valeraldehyde (VA) has been studied in two hepatoma cell lines, the rat HTC and mouse Hepa 1c1c7 cells. The cytotoxicity of the four aldehydes to these two cell lines has been compared. The end-points for evaluating cytotoxicity were 1) total macromolecular content (TMC) of confluent cultures, and 2) colony forming ability of dividing cells. These two assay systems had different sensitivities for the toxicity of aldehydes, probably due to different numbers of target cells. The activities of aldehyde dehydrogenases (NAD- and NADP-dependent, ALDH), alcohol dehydrogenase and aldehyde reductase were markedly greater in the HTC cell line compared to the Hepa 1c1c7 cell line, especially with BA as substrate. The cytotoxicities of aldehydes were generally stronger in the HTC cell line than in the Hepa 1c1c7 cell line; with the CF test. Particularly, BA was highly toxic to the HTC cells, which possessed the highest ALDH levels. Moreover, the treatment with (diethylamino)benzaldehyde, an ALDH inhibitor, completely abolished the toxicity of BA. Taken together, all these findings suggest that several cell lines expressing different aldehyde metabolizing activities could be used especially in the pre-screening phase to distinguish the metabolism-dependent cytotoxic effects from the metabolism independent effects.

A comparative study of leukaemia inhibitory factor and interleukin-1α intracellular content in a human keratinocyte cell line after exposure to cosmetic fragrances and sodium dodecyl sulphate

According to European laws animal testing in cosmetic industry will be prohibited in a few years and it will be replaced by alternative methods based on cell and tissue culture. Many ingredients of cosmetic formulations are potentially causes of skin inflammation and sensibilization. Since cytotoxicity is known, among other factors, to trigger irritation, in an alternative model for evaluation of skin irritation, it can be considered also the precocious release of inflammatory mediators, i.e. cytokines, originating mainly from keratinocytes. In this in vitro study we have analysed some parameters directly or indirectly related to irritation/inflammation, in NCTC 2544 human keratinocytes during short-time exposure to some potential irritants cosmetic fragrances, included in the European Laws 2003/15/EEC. IIC50 was extrapolated by MTT and NRU viability indexes after exposure of cell ultures to Geraniol Limonene and Benzylic Alcohol for 1, 3 and 6h. NCTC cells were then exposed to sub-toxic doses of selected compounds and interleukin-1alpha (IL-1alpha) and leukaemia inhibitory factor (LIF) expressions were analysed as early proinflammatory cytokines. To our knowledge our findings demonstrated for the first time that NCTC cells synthesize and modulate LIF after exposure to selected irritating stimuli. Moreover, our results give evidence on LIF role as in vitro precocious endpoint for the assessment of the risk in cosmetic field, because its response under irritation stimuli is very quick and comparable to IL-1alpha.

Use of established hepatoma cell lines in biotoxicology

ConclusionsIn conclusion, hepatoma cell lines displaying multiple combinations of bioactivating and inactivating enzymes, even if do not seem to represent liver-specific bio-indicators of toxicity, are a polyvalent liver system, suitable to evaluate particular end-points.In conclusion, hepatoma cell lines displaying multiple combinations of bioactivating and inactivating enzymes, even if do not seem to represent liver-specific bio-indicators of toxicity, are a polyvalent liver system, suitable to evaluate particular end-points.

Conscientious Objection to Animal Experimentation in Italian Universities

Simple Summary This paper examines the trend of Italian academic faculties in complying with the obligation to inform university students of their right to exercise their conscientious objection to scientific or educational activities involving animals, hereafter written as “animal CO”, as established by Law 413/1993, “Norme sull’obiezione di coscienza alla sperimentazione animale” (“Rules on conscientious objection to animal experimentation”), thereafter “Law 413/1993”. Despite an increasing interest in the principles of animal ethics by the international community, this law is still largely disregarded more than 20 years after its enactment. The Ethics Committees, Animal Welfare Committees, as well as the Italian Ministry of Education, University and Research should preside over and monitor the Universities’ compliance with the duty to disclose animal CO. Abstract In Italy, Law 413/1993 states that public and private Italian Institutions, including academic faculties, are obliged to fully inform workers and students about their right to conscientious objection to scientific or educational activities involving animals, hereafter written as “animal CO”. However, little monitoring on the faculties’ compliance with this law has been performed either by the government or other institutional bodies. Based on this premise, the authors have critically reviewed the existing data and compared them with those emerging from their own investigation to discuss limitations and inconsistencies. The results of this investigation revealed that less than half of Italian academic faculties comply with their duty to inform on animal CO. Non-compliance may substantially affect the right of students to make ethical choices in the field of animal ethics and undermines the fundamental right to express their own freedom of thought. The Italian Ministry of Education, Universities and Research, ethics committees and animal welfare bodies should cooperate to make faculties respect this law. Further research is needed to better understand the reasons for the current trend, as well as to promote the enforcement of Law 413/1993 with particular regard to information on animal CO.

Quinolone/fluoroquinolone susceptibility in Escherichia coli correlates with human polymicrobial bacteriuria and with in vitro interleukine-8 suppression

Urinary tract infections (UTIs) are frequently polymicrobial diseases mainly sustained by Escherichia coli in association with other opportunistic pathogens. Cystitis and pyelonephritis are usually accompanied by an inflammatory response, which includes neutrophil recruitment. Uropathogenic E. coli possess the ability to evade host defenses, modulating the innate immune response. The aim of this study was to determine whether particular E. coli strains correlate with polymicrobial bacteriuria and whether escape from the early host defenses and microbial synergy could lead to mixed UTIs. We evaluated 188 E. coli-positive urine samples and assessed the relationships among polymicrobism, neutrophil presence and several traits of E. coli isolates (virulence factors such as hlyA, fimA, papC and their relative products, i.e. hemolysin, type 1 and P fimbriae, and cnf1, their phylogenetic group) and their ability to suppress cytokine response in 5637 bladder epithelial cells. Escherichia coli susceptibility toward quinolones and fluoroquinolones, known to be linked to the pathogenicity of this species, was also considered. We found significant correlations among polymicrobial bacteriuria, absence of pyuria and quinolone/fluoroquinolone susceptibility of E. coli isolates and their enhanced capability to suppress interleukin-8 urothelial production when compared with the patterns induced by the resistant strains.

Alternative approach to animal testing and cell cultures, according to European laws

ALTEX 34(3), 2017 441 such as skin/eye irritation and partially for skin sensitization. Some non-validated alternative approaches, such as read-across and weight-of-evidence approaches, are also accepted in the EU when sufficiently justified. The OECD launched a program on the development of Adverse Outcome Pathways (AOP) in 2012. AOP is an analytical construct that starts from a mechanistic/ cellular/in vitro sub-profiling to reach the effects observed in populations, and describes sequential events at different levels of biological organization that lead to adverse health or ecotoxicological effects. Dr Stefania Vernazza (LARF-DIMES, University of Genoa) provided an overview on in vitro 3D alternative methods for evaluation of the human risk related to exposure to chemical compounds. International organizations, such as ECVAM and OECD, cooperate on the development of regulations on reducing the use of laboratory animals through the validation of in vitro toxicity tests. Some OECD test guidelines (439, 431 and 492) accept data from in vitro 3D models to classify a substance as irritant/corrosive for skin or eye without the need to perform animal tests. 3D tissues for these applications are commercially available. Prof. Anna Maria Bassi (LARF-DIMES, University of Genoa) explained the concept of a reliable predictive toxicology. Most of the failures in drug discovery are a result of the low predictivity of preclinical animal models for human diseases such as autism syndrome or asthma. There is the pressing need for new toxicity testing models based on human biology. In this regard, Dr Bassi spotlighted the development of a mini-brain by Thomas Hartung’s team (CAAT, Johns Hopkins University) to improve research and drug development for neurodegenerative diseases. Mini-brains derive from patient cells and so can reproduce epigenetic interactions. The second part of the lecture was focused on AOPs, which provide a comprehensive knowledge of human disease and link chemical properties of a toxicant, its macro-molecular interactions, cellular/organ/organism responses and adverse outcomes in the population. This complex of data is critical to identify exposure biomarkers and toxicity pathways. The OECD’s AOP website invites project proposals, offers stakeholder summaries, proposal templates and guidance on developing AOPs. The AOP program foresees a biannual update for new project proposals and new information on ongoing projects. Dr Giorgio Mattei (Research Centre “E. Piaggio”, University of Pisa, Italy) presented an update on innovative technologies for dynamic in vitro 3D models. He explained the characteristics of biomimetic in vitro models resulting from combinations of cell culture systems, advanced materials design, tissue engiOn September 29-30, 2016, the 8th edition of the training course on “Alternative Approach to Animal Testing and Cell Cultures, According to European Laws”, was held in Genoa, chaired by Prof. Anna Maria Bassi and organized and hosted by the team of Laboratory Analysis and Research in Physiopathology (LARF), Department of Experimental Medicine, University of Genoa, Italy. The course was focused on practical work and demonstration/lessons by specialist(s) aiming to supply basic knowledge or improve existing knowledge on alternative methods. Each edition of the course provides an update on innovative in vitro models with a particular emphasis on 3D models. Substantial practical time is included to allow participants to gain hands-on experience. Twenty-four participants, from experts of in vitro methods to undergraduates, from all over Italy took part. The course was opened by Prof. Giovanni Murialdo and Prof. Adriana Voci, coordinators of the degree courses in Medicine and Surgery, and Biological Sciences, respectively, at the University of Genoa. Both stressed the need for such events to ensure that research in Italy for the study of risks to human and animal health following exposure to chemical compounds (drugs, pollutants, food additives, etc.) is in step with new technologies. Prof. Rosagemma Ciliberti (DISSAL, University of Genoa) and Susanna Penco (LARF-DIMES, University of Genoa) highlighted ethical issues of animal-based research. According to Italian Law 413/1993 on “Rules on conscientious objection to animal experimentation”, physicians, researchers, students and healthcare providers can choose not to take part in activities that involve animal experimentation. Conscientious objection to animal experimentation arose from the acknowledgement of animals as conscious beings. Dr Chiara Scanarotti (LARF-DIMES, University of Genoa) gave an update on the development and validation of several well-standardized and reproducible alternative methods for toxicity testing of chemical compounds, underlining how the use of human instead of animal cells improves the accuracy and relevance of the in vitro tests. The lecture highlighted 3D models, spheroids, body-on-a-chip and the latest advancements in stem cell research. Dr Costanza Rovida (CAAT Europe – University of Konstanz, Germany) presented a lecture on the implementation of alternative methods. Full replacement of in vivo tests is a lengthy process as they are mandatory for the registration and risk assessment of chemicals (drugs, additives, etc.) and the use of alternative methods requires formal validation before acceptance. To date, in vitro tests are accepted for some endpoints, Alternative Approach to Animal Testing and Cell Cultures, According to European Laws

Modulation of CYP1A1 by PKC Inhibitors and TPA Pre-Treatments in MH1C1 Rat Hepatoma Cells Exposed to 3 -Methylcholanthrene

Cytochrome P4501A1 (CYP1A1), an enzyme known to metabolize polycyclic aromatic hydrocarbons, is regulated by the aryl hydrocarbon receptor (AhR). The involvement of protein kinase C (PKC) in the regulation of AhR signal transduction pathway, has been widely studied but the role of specific PKC isoform(s) involved in this process it is not well clarified. To study which PKC isoform(s) is implicated in the regulation of CYP1A1, in the poorly tumorigenic MH1C1 rat hepatoma cells, we examined the effects of some PKC pharmacological inhibitors, Calphostin C (CAL), Staurosporine (STA) and H7, and of 12-0-tetradecanoyl phorbol 13-acetate (TPA), a PKC activator, on basal and 3- methylcholanthrene (MC)-induced CYP1A1 protein expression and mediated ethoxyresorufin O-deethylation (EROD) activity. In parallel, the activities of PKC-α, -βI, -δ and -e isoforms, the most expressed in MH1C1 cells, were monitored. After pre-treatment with CAL, STA and H7, the MC-induced CYP1A1 protein and EROD activity were rapidly reduced with temporal profile similar to the profile of the activity of α and β1 PKC isoforms. Moreover, TPA pre-treatment induced a biphasic effect on EROD activity, and a decline of PKC -βI and -α, in first instance, and -δ and -e activities later on. These findings clearly show that, in MH1C1 cells, PKC is involved in CYP1A1 regulation and that α and βI classic PKC isoforms play an active role in modulating this process.

Susceptibility of Hepatoma Cells to Lipid Peroxidation and Adaptation of ALDH 3C Activity to Iron-Induced Oxidative Stress

Free rcal iron-induced oxidative stress causes membrane lipid peroxidation and conseqt cell damage (Bacon et al., 1985), a process that, in rat liver, is significantly lower tumor than normal tissue (Dianzani et al., 1984, 1986; Borrello et al., 1985). Factors contriing to this difference include a reduction in cytochrome P450 (an enzyme systemplicated in the production of initiating and propagating radicals), a reduction in the peidizable substrate arachidonic acid (ARA), an increase in the lipid soluble anti- oxidan-tocopherol (Borrello et al., 1985; Cheeseman et al., 1988).