B[a]P can cause several health issues in fish, such as liver toxicity, genetic mutations, and cancer. The effects of lower B[a]P concentrations on genotoxicity, tumor formation, and several cancerous traits were tested in liver cells, which could trigger tumor growth in edible marine sparids. This study presents the advancements of an in vitro tumor model that includes multiple liver cell types, generated by asymmetric nuclear divisions in mutated, heterogeneous, malignant liver stem cells (LSC) from tumor foci of cultured sea bream hepatocytes, chronically exposed to the toxic harmful B[a]P at low concentrations. Primary cultured hepatocytes from Sparus aurata (SaHeP) were initially exposed to benzo[a]pyrene (B[a]P) at concentrations ranging from 100 & micro;g/mL to 1 pg/mL to establish dose-response curves. Subsequently, the concentration range was refined to 1 & micro;g/mL and below 1 ng/ mL (0.1, 0.01, and 0.001 ng/mL) for 24 and 72 h. Exposure was then extended to four days using concentrations from 0.5 ng/mL to 0.5 pg/mL (0.5, 0.05, 0.005, and 0.0005 ng/mL). Cell viability biomarkers, specifically MTT and Neutral Red (NR), were utilized to assess cytotoxic response curves and identify the sub-lethal thresholds for investigating tumorigenesis. We characterized nuclear atypia, DNA damage, and cytostructural alterations in both hepatocytes and liver stem cells using markers including PCNA (proliferation), Caspase-3, Annexin V, CFDA, FITC-phalloidin, CK18, and albumin. These parameters were evaluated via morphological, immunocytochemical, and immunofluorescence analyses to elucidate the mechanisms of cancer progression. The findings revealed an unexpected role for activated caspase-3; it was predominantly expressed in rapidly dividing oval cells (OVCs), where it appeared to confer resistance to apoptosis, thereby facilitating proliferation and neoplastic transformation. Following 72 h of exposure to 1 & micro;g/mL B[a]P, liver progenitor cells (LPCs) expanded, forming germinal centers characterized by a core of replicating OVCs. These clusters of mutated CSCs eventually developed into large malignant aggregates of hepatocellular carcinoma (HCC). At concentrations below 1 ng/mL, smaller CSC populations formed aggressive clones that exhibited metastatic traits, including the loss of contact inhibition and increased invasive capacity. We identified large, activated Hepatic stellate cells (HSCs) that began to spread invasive, multinucleated protrusions and displayed infiltrating surface extensions, typical of malignant carcinomas. The tumor-driven proliferation of aggressive LSC clones, caused by dysregulated self-renewal, leads to highly proliferative tumor masses. The current findings showed that the cells couldn't recover from chronic, persistent B[a]P-induced damage, even at the lowest tested concentrations, leading to irreversible and invasive lesions.
Liver stem cell tumorigenesis in gilthead sea bream induced by B[a]P toxicity: In vitro insights.
Santacroce M.
Writing – Original Draft Preparation
;Pugliese N.Investigation
;Leone R.Methodology
;Cordisco M.Methodology
;Passantino G.Formal Analysis
;Zizzo N.Supervision
;Tinelli A.Writing – Review & Editing
2026-01-01
Abstract
B[a]P can cause several health issues in fish, such as liver toxicity, genetic mutations, and cancer. The effects of lower B[a]P concentrations on genotoxicity, tumor formation, and several cancerous traits were tested in liver cells, which could trigger tumor growth in edible marine sparids. This study presents the advancements of an in vitro tumor model that includes multiple liver cell types, generated by asymmetric nuclear divisions in mutated, heterogeneous, malignant liver stem cells (LSC) from tumor foci of cultured sea bream hepatocytes, chronically exposed to the toxic harmful B[a]P at low concentrations. Primary cultured hepatocytes from Sparus aurata (SaHeP) were initially exposed to benzo[a]pyrene (B[a]P) at concentrations ranging from 100 & micro;g/mL to 1 pg/mL to establish dose-response curves. Subsequently, the concentration range was refined to 1 & micro;g/mL and below 1 ng/ mL (0.1, 0.01, and 0.001 ng/mL) for 24 and 72 h. Exposure was then extended to four days using concentrations from 0.5 ng/mL to 0.5 pg/mL (0.5, 0.05, 0.005, and 0.0005 ng/mL). Cell viability biomarkers, specifically MTT and Neutral Red (NR), were utilized to assess cytotoxic response curves and identify the sub-lethal thresholds for investigating tumorigenesis. We characterized nuclear atypia, DNA damage, and cytostructural alterations in both hepatocytes and liver stem cells using markers including PCNA (proliferation), Caspase-3, Annexin V, CFDA, FITC-phalloidin, CK18, and albumin. These parameters were evaluated via morphological, immunocytochemical, and immunofluorescence analyses to elucidate the mechanisms of cancer progression. The findings revealed an unexpected role for activated caspase-3; it was predominantly expressed in rapidly dividing oval cells (OVCs), where it appeared to confer resistance to apoptosis, thereby facilitating proliferation and neoplastic transformation. Following 72 h of exposure to 1 & micro;g/mL B[a]P, liver progenitor cells (LPCs) expanded, forming germinal centers characterized by a core of replicating OVCs. These clusters of mutated CSCs eventually developed into large malignant aggregates of hepatocellular carcinoma (HCC). At concentrations below 1 ng/mL, smaller CSC populations formed aggressive clones that exhibited metastatic traits, including the loss of contact inhibition and increased invasive capacity. We identified large, activated Hepatic stellate cells (HSCs) that began to spread invasive, multinucleated protrusions and displayed infiltrating surface extensions, typical of malignant carcinomas. The tumor-driven proliferation of aggressive LSC clones, caused by dysregulated self-renewal, leads to highly proliferative tumor masses. The current findings showed that the cells couldn't recover from chronic, persistent B[a]P-induced damage, even at the lowest tested concentrations, leading to irreversible and invasive lesions.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
