Section of Toxicology and Risk Assessment Department of ... · Section of Toxicology and Risk...

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