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Section of Toxicology and Risk Assessment Department of Pharmacological and Biomolecular Sciences (DiSFeB),
University of Milan.-Italy
Porta hepatis
Hepatic artery
Portal vein
right lobe left lobe
Hepatic vein
Bile canaliculus
Sinusoid
• HEPATOCELLULAR DEGENERATION AND INTRACELLULAR ACCUMULATION
– Ballooning and foamy degeneration
– Steatosis
• Necrosis and Apoptosis
– Zonal necrosis
– Massive necrosis
• Inflammation (hepatitis)
• Regeneration
• Fibrosis – Cirrhosis
tRNA
RNA
DNA
VLDL
TG PLipids Protein
FATTY ACIDS
SYNTHESIS
DIET LIPIDS
TISSUE DEPOSIT
mRNA
• HEPATOCELLULAR DEGENERATION AND INTRACELLULAR ACCUMULATION
– Ballooning and foamy degeneration
– Steatosis
• NECROSIS AND APOPTOSIS
– Zonal necrosis
– Massive necrosis
• Inflammation (hepatitis)
• Regeneration
• Fibrosis – Cirrhosis
• Lipid peroxidation (chlorinated solvents)
• Mitochondrial damage
• Deregulation of cytoskeletal proteins
• Massive increase of the intracellular Ca2+
• Covalent binding to macromolecules (methionine, amanita, galactosamine, DMNA)
• Necrosis
(octapeptide)
Na+ taurocolato peptide carrier
Na+ taurocolate carrier peptide
(octapeptide)
Na+ taurocolato peptide carrier
(octapeptide)
α Amanitin
m RNA
Protein syntesis NECROSIS
Na+ taurocolato
(octapeptide)
Na+ taurocolato peptide carrier
(octapeptide)
α Amanitin
m RNA
Protein syntesis NECROSIS
Na+ taurocolate carrier peptide Na+ taurocolato
• Repeated exposure
– Fe (ferritine in lysosomes)
– Cu (metallothioneine in lysosomes)
• oxidation reactions with formation of reactive oxygen species (ROS)
• reactions of lipid peroxidation ( pO2)
– ethanol, alothane
• Immune reaction
• Adduct formation between xenobiotics and blood
proteins after repeated exposure
• Formation of antibodies
– If antibodies are located on the membrane of hepatocytes,
cytolytic effect Alothane Diclofenac Acetaldehyde
MORPHOLOGICAL PATTERNS OF HEPATIC INJURY
• Hepatocellular degeneration and intracellular accumulation
– Ballooning and foamy degeneration
– Steatosis
• Necrosis and Apoptosis
– Zonal necrosis
– Massive necrosis
• INFLAMMATION (HEPATITIS)
• Regeneration
• Fibrosis – Cirrhosis
MCP-1
CCl4
Acetominofene
1,2-diclorobenzene MCP-1 LPS
CCl4
Acetominofene
1,2-diclorobenzene MCP-1 LPS
CCl4
Acetominofene
1,2-diclorobenzene MCP-1 LPS
Vit A
MORPHOLOGICAL PATTERNS OF HEPATIC INJURY
• Hepatocellular degeneration and intracellular accumulation
– Ballooning and foamy degeneration
– Steatosis
• Necrosis and Apoptosis
– Zonal necrosis
– Massive necrosis
• Inflammation (hepatitis)
• REGENERATION
• Fibrosis – Cirrhosis
After a 75% ablation of its total mass, liver only needs 4 months to regain its initial weight. A liver in good health works as efficiently at 80 years of age as it did at the age of 20.
• CYTOLYSIS (alteration of the actin filaments)
– Microcystins (Microcystis aeruginosa) • Acutely: disruption of cytoplasmic membranes, and
• Chronically: activation of oncogenes that trigger gastrointestinal epithelial tumors
• IRREVERSIBLE DAMAGE – vinyl chloride (angiosarcoma)
– arsenic (haemangiosarcoma)
– thorium dioxide or thorotrast (angiosarcoma)
• Hepatocellular degeneration and intracellular accumulation
– Ballooning and foamy degeneration
– Steatosis
• Necrosis and Apoptosis
– Zonal necrosis
– Massive necrosis
• Inflammation (hepatitis)
• Regeneration
• FIBROSIS
– Cirrhosis
• DIRECT CYTOLYTIC DAMAGE
• CHRONIC INFLAMMATORY PHENOMENON
Deposition of fibrous tissue (collagen fibers) in middle hepatic vein or portal tract
Scar tissue (collagen slices)
Not reversible
Survival prognosis: poor Etanolo cronico
Arsenico
Vitamina A (danno delle cellule di Ito)
• Cytotoxic Injury
• Disturbance of hepatic function
• Cholestatic Injury
• Cancerogenicity
1-2%
Peripheral neuropathy Paresthesia
Ataxia
Liver
Isonicotinic ac.//acetylhydrazine CYP
1-2%
N-acetyltransferase
Peripheral neuropathy Paresthesia
Ataxia
Liver
Isonicotinic ac.//acetylhydrazine CYP
Hepatotoxicity 1 – 2%
carbocatione
carbocatione
carbocatione
DNA alkylation
52%
42%
52%
42%
52%
42%
52%
42%
Mercapturic acid
52%
42%
Glutathione 30%
52%
42%
Mercapturic acid
Therapy: N – Ac- Cys
• Cytotoxic Injury
• Disturbance of hepatic function
• Cholestatic Injury
• Cancerogenicity
UDP-glucuroniltransferasi (UGT)
induction
toxicity
Protein reactive
Cocaina
CYP2B
Cocaine
100 1000
CYP2B
• Cytotoxic Injury
• Disturbance of hepatic function
• Cholestatic Injury
• Cancerogenicity
• ALTERED COMPOSITION OF BILE
– elevated serum concentrations of endogenous bile compounds (bile salts and bilirubin
jaundice
• VOLUME REDUCTION AND BILIARY FLOW
– (phalloidin, Colchicine, Mn)
Phalloidin ( actin polim.)
Colchicine ( tubulin polymerization)
NECROSIS
Proteins
Bile acids
BILE CANALICULUS
• MECHANICAL OBSTRUCTION OF BILE DUCT
– extrahepatic formation of calculi (cholelithiasis)
• internal viscosity canaliculi
• concentrations and cholesterol crystallization
• precipitation of calcium salts of bilirubin caused by infectious biliary tract
– EXTRAHEPATIC OVERPRESSURE PHENOMENA
– Pancreatic cancer
– Proliferation of the bile duct
– Focal necrosis of the liver parenchyma
UDP-glucuroniltranferasi (UGT)
50x
excretion
excretion
50x
Multidrug Resistance
Associated Protein Mrp2
excretion
50x
5.000x Multidrug Resistance
Associated Protein Mrp2
excretion
50x
JAUNDICE
5.000x Multidrug Resistance
Associated Protein Mrp2
BILE
FIBRATI
Multi Drug Resistence Protein
FIBRATES
Myotoxicity Myoglobinuria Renal failure
AUC Cerivastatin
FIBRATES
• Cytotoxic Injury
• Cholestatic Injury
• Disturbance of hepatic function/clearance
• Carcinogenicity
Carcinogen
Procarcinogen (non reactive)
Carcinogen + DNA
Promoting agent
Cancer
Mutation/Initiation Mutation
Promotion
Cancer
• CARCINOGENS NON-GENOTOXIC
• Substances that cause chronic damage (necrosis)
• Substances that cause mitochondrial, peroxisome proliferation (DEHP)
• Marked proliferation and hyperplasia of the liver (Phenobarbital)
• Substances that induce Mixed Functions Oxidase (MFO - CYP450)
• Strong promoters genotoxic carcinogens (TCDDs, PCBs, controlled by
Aryl Hydrocarbon receptor (AhR).
57
• CARCINOGENIC NON-genotoxic
• Hormones
– Increased mitotic activity,
– Promoters (activated genotoxic carcinogens)
– (Oral contraceptives, ethinyl-estradiol etc.)
• Other components
– High-caloric diet (lipids)
– Diets deficient in choline (DNA hypomethylation)
Key events
1. Metabolism
2. Interaction with Nuclear
DNA
3. Mutation (production of an initiated cell)
4. Selective clonal expansion of
the initiated cell
5. Neoplasm formation
Examples
Aflatoxin B1 (AFB)
Naphthylamine
2-Acetylaminofluorene (AAF)
N–methyl–N–nitrosourea (MNU)
Ethyl methanesulfonate (EMS)
O O
O
O O
O O O
O
O O
O
O CYP1A2
CYP3A4
O
HO
Glutatione-S- O
HO
HO
H O 2 GSH
GST
Legame Covalente
DNA
N
UDP-glucuroniltranferasi
Key events
1 Hepatocellular Necrosis
2. Induction of cell proliferation
3a. Formation of initiated cell
3b Selective clonal expansion of the initiated cell
4. Neoplasm formation
Examples
Carbon tetrachloride
Hexachlorobenzene (HCB)
Polychlorinated Biphenyls
(PCBs)
C C l4
C Y P 2 E 1
e 3C C l
O2C C l O O
3
C H C l3
+ C l
_R H
R
C l o r o f o r m i o
phosgene
2HCl + CO2
Trichloromethyl peroxide radical
Key events
1 Binding of compound or metabolite to receptor
2. Induction of cell growth gene expression
3 Selective clonal expansion of the initiated cell
4. Neoplasm formation
Examples
Constitutive androstane receptor (CAR)
Peroxisome proliferator-activated receptor alpha (PPARa)
Estrogen
Aryl hydrocarbon receptor (Ahr)
P
Retinoid Receptor X
Phenobarbital-Responsive Enhancer Module
PHENOBARBITAL
P
P
P Costitutively Active Receptor (CAR)
P
car
car
car RXR
66
Peroxisome proliferator Activated Receptor-
PEROXISOME (metabolism of fatty acids)
67
Peroxisome proliferator Activated Receptor-
Mitogen-activated protein kinase
Key events
1. Induction of ROS (endogenous or exogenous)
2 Formation of oxidized DNA, protein or lipid products
3. Modification of cell growth gene expression
3 Selective clonal expansion of the initiated cell
4. Neoplasm formation
Examples
Chlorinated compounds Dieldrin, DDT
Inflammation hepatitis
Metal overload FE, CU
•Organochloride Insecticide
•Not mutagenic,
•Non genotoxic
•Selectively induces liver tumors in mice
O
Cl
Cl
Cl
Cl Cl Cl
PRODUCES A DOSE DEPENDENT INCREASE IN OXIDATIVE STRESS
LIPID PEROXIDATION
All Basophilic Eosinophilic 0
1
2
3
4
5
6
7
untreated
vitamin E
dieldrin
dieldrin + vitamin E
Foci Phenotype
Rela
tive
Vol
ume o
f H
epa
tic
Foc
i
Endogenous Sources of ROS Exogenous Sources of ROS mitochondria radiation cytochrome P450 ozone peroxisomes hyperoxia inflammatory cells xenobiotics
Oxidative Damage lipid, DNA, protein
OH· = hydroxyl radical
NO· = nitrile radical O2
− = anion superoxide
ROO = peroxy radical
H2O2
Fe(II)
Fe(III)
OH + OH
H2O2
O2
OH + OH + O2
✚
Reazione di Fenton
Reazione di Haber Weiss
H2O
OH + H
Stadi intermedi
, anion / superoxide radical; OH , hydroxyl radical; H2O2, hydrogen peroxide; O2
1. ENZYMATIC SYSTEMS.
eg .: catalase, superoxide dismutase
2. ENDOGENOUS ANTIOXIDANTS.
eg. Glutathione
3. ANTIOXIDANTS ENDOGENOUS / EXOGENOUS.
eg. vitamins (A, C, E)
, anione/radicale superossido; OH , radicale idrossilico; H2O2, perossido d’idrogeno; SOD, superossido dismutasi; CAT, catalasi; GPO, glutatione perossidasi.
O2
H2O2
2GSH 2GSSG
2H2O
H2O2
2H2O Fe(II)
Fe(III)
O2
O2+e
O2
O2
2H +
OH + OH
O2
1. ENZYMATIC SYSTEMS.
eg .: catalase, superoxide dismutase
2. ENDOGENOUS ANTIOXIDANTS.
eg. Glutathione
3. ANTIOXIDANTS ENDOGENOUS / EXOGENOUS.
eg. vitamins (A, C, E)
O2
H2O2
2GSH 2GSSG
2H2O
H2O2
2H2O Fe(II)
Fe(III)
O2
O2+e
O2
2H +
OH + OH
O2
GSH
GSH GSSG
NADPH NADPH-
Endogenous Sources of ROS Exogenous Sources of ROS mitochondria radiation cytochrome P450 ozone peroxisomes hyperoxia inflammatory cells xenobiotics
Oxidative Damage lipid, DNA, protein
OH· = hydroxyl radical
NO· = nitrile radical O2
− = anion superoxide
ROO = peroxy radical
Antioxidants enzymatic • SOD, CAT, GSH perox non-enzymatic • VitE, GSH, VitC
CCl4 CCl3
OOCCl3
CCl4 CCl3
OOCCl3
NECROSIS
PROXIMAL TUBULE
filtration and reabsorption site chromium, mercury, lead, cadmium, antibiotics, PCBs
amino acids, glucose in the urine (not absorbed by the blood)
DISTAL TUBULE
Na +, K +, and H + secretion site, amphotericin B (antifungal)
decreased urine acidification
ENDOSOME
LySOSOME aa
ENDOSOME
LYSOSOME
ALBUMIN PROTEINURIA
VASCULAR EFFECT TUBULAR EFFECT
renal blood flow obstruction
glomerular filtration rate (GRF)
filtration pressure
reflux
PREDOMINANT RISK FACTORS FOR NSAID –ASSOCIATED ACUTE
RENAL FAILURE (ARF) REQUIRING HOSPITALIZATION
Male older than 65
High drug dose
Cardiovascular diseases
Recent hospitalization for non renal diseases
Concomitant use of other potentially nephrotoxic drugs
Tubular-glomerular feedback Further constriction of arterioles
Constriction of the afferent arterioles leading to decrease
of glomerular filtration
Tubular-glomerular feedback Further constriction of arterioles
glomerular hydrostatic pressure
glomerular plasma flow glomerular plasma flow
constriction
NSAIDs
Glomerular filtration
Intralobular pressure
ischemia
glomerular hydrostatic pressure
Glomerular filtration
Intralobular pressure
CADMIUM
CADMIUM
glomerular plasma flow glomerular plasma flow
constriction ischemia
• Concentration of urea and plasma creatinine
• Concentration of organic anions in urine
– N-methyl-nicotinamide (NMN) or triethylammonium (TEA).
• Glomerular filtration
– Clearance of the polysaccharide inulin
• Renal blood flow
– Clearance and excretion of p-ac aminoippurico (PAH)
Clearance: assay for measuring the efficiency of kidney excretion based on the amount of blood "cleaned up" by a substance in a minute Substance in the blood / substance excreted in the urine / fixed time
• Changes in urine volume
• Osmolarity of urine
• urinary pH (usually acid)
• Excretion of electrolytes, Na + and K +
• Proteins – low molecular weight: tubular damage
– high molecular weight (> 60,000): glomerular injury
• Glucose
• Specific enzymes – (N-acetyl- -D-glucosaminidase, glutamyltransferase)
Prostaglandin Endoperoxidase
Synthetase (PES)
N-acetil p-benzochinonimmina p-benzochinonimmina
PROTEINS
MEDULLARY
CSP
CSP
CSP
CSP
Organic Anion Transporter1 (OAT1)
Organic Cation Carrier
Blood
Proximal tubule cell
CSP Blood
e-
Proximal tubule cell
CSP
CSP
CSP
Organic Anion Transporter1 (OAT1)
Organic Cation Carrier
CSP
Probenecid nefrotossicità
Blood
Cellula Tubulo Prossimale
CSP
CSP
CSP
Organic Anion Transporter1 (OAT1)
Organic Cation Carrier
CSP
Probenecid nefrotossicità
Mepifenidolo nefrotossicità
Blood
Proximal tubule cell
CSP
CSP
CSP
Organic Anion Transporter1 (OAT1)
Organic Cation Carrier
AG
AG
Blood
Proximal tubule cell
Fosfoinositoli
Lumen
AG
AG
AG
Fosfoinositoli
Blood
Proximal tubule cell
Lumen
AG
AG
AG
Fosfoinositoli
LISOSOMA AG
Metabolism phospholipid
Blood
Proximal tubule cell
Lumen
AG
AG
AG
Fosfoinositoli
AG
MITOCHONDRION ROS
NECROSIS Metabolism phospholipid
LISOSOMA
Blood
Proximal tubule cell
Lumen
• Tetracicline
– tubulo prossimale
• poliuria, glicosuria, aminoaciduria
• Penicilline e sulfamidici
– nefrite infiammatoria interstiziale
• Antifungini
– nefrone
• azotemia, poliuria, ipocaliemia, acidosi tubulare, vasocostrizione arteriolare, legame colesterolo membrane permeabilita’ membrana
• Tetracyclines – proximal tubule
• polyuria, glycosuria, aminoaciduria
• Penicillins and sulfonamides – inflammatory interstitial nephritis
• Antifungals – nephron
• azotemia, polyuria, hypokalemia, tubular acidosis, arteriolar vasoconstriction, bonding membrane cholesterol membrane permeability
CHEMOTHERAPICS
• Reaction with thiol groups (glutathione, cysteine, methionine)
• Inhibition of DNA synthesis and protein
• Proteinuria
• Enzimuria
• Polyuria
• Loss brush border of the proximal convoluted tubule cells (Day 2)
– Focal necrosis distal convoluted tubules
– Focal necrosis collecting duct
ENVIRONMENTAL
NEPHROTOXIC AGENTS
• Proximal tubule (chronic exposure)
2 Globulin 2 Globulin TCET
Glomerular filtration Resorption of complex proximal tubule cells Internalization lysosomal Lysosomal degradation (slow in the rat) Necrosis Cell hyperproliferation compensatory Neoplasia (only male rat)
2 Globulin DCB
tert - Buthyl Alcohol
rat human
2 Globulin ter-butyl alcohol
renal neoplasms
Bromphenol
Bromo Hydroquinone (BHQ)
2-bromodiglutatione-S-etilidrochinone
N-acetil-cisteina-BHQ
2-bromomonoglutatione-S-etilidrochinone
glutathione
P450
(Proximal tubule- liasi)
oxidation
Kidney
Liver
BROMOBENZENE
liasi Reactive thiol
KIDNEY (Balkan endemic nephropathy)
Necrosis Fibrosis
Interstitial sclerosis Urinary tract tumors
Formation of free radicals Production of prostaglandins Intracellular alkalinization
• Low doses: the proximal tubule
• High doses: also distal convoluted tubule
• Vasoconstriction
• Bond with thiol groups (Cys-Cys, enzymes and proteins)
• Loss brush border and dissolution of cell membranes
• Necrosis
Polyuria - albumin
Uptake proximal tubule cells
Interaction with protein -SH group
Proliferation endoplasmic reticulum
Altered mitochondria (oxidative stress)
cytoplasmic Ca
Cytoplasmic vacuolization
Agglutination nuclear chromatin
Cytoplasmic membrane rupture
Necrosis
• Proximal tubule (chronic exposure)
metallothionein Cd2+-metallothionein
100 e 200 µg Cd2+/g Cd uptake by the proximal tubule cells intracellular thiols (also mitochondrial) Lysosomal degradation Proximal tubule necrosis Ca ++ urine kidney stones Dysfunction of the distal tubule
(6.5kD)
• Proximal convoluted tubule (reversible acute dysfunction)
• Cytomegaly and karyomegaly (binding to nuclear proteins)
• Swelling of mitochondria
– damage of respiratory chain
• Atrophy tubules
• Glomerular sclerosis
• Poor sodium reabsorption, glucose, amino acids and phosphates
• Inhibition of heme synthesis
• Experimental neoplasms
• Cr6+
– Proximal convoluted tubule
– glucose reabsorption. Glycosuria
– Loss brush border cell membrane proximal
convoluted tubule
– Necrosis proximal convoluted tubule cells
• CCL4
Phosgene
C C l4
C Y P 2 E 1
e 3C C l
O2C C l O O
3
C H C l3
+ C l
_R H
R
C l o r o f o r m i o
PROTEINS
DNA