Secado de pulpa de plátano y de manzana mediante bomba de calor

Toxicant chemicals enter plants through foliar uptake or root uptake and can bind to waxes on leaf surfaces. The rate of movement and target sites depends on phys- iocochemical properties of the toxicant (Henry’s law constant, hydrophilicity) and environmental conditions including temperature, humidity, exposure to UV light, and height of the leaf boundary layer.

Figure 3.3 Henderson-Hasselbach equations – the relationship between

Stomatal uptake of pollutants in terrestrial vascular plants can occur in the gaseous form and through both wet and dry deposition for sulphur dioxide (SO2), carbon dioxide (CO2), carbon monoxide (CO), oxides of nitrogen (NOx), ammonia (NH3), hydrogen peroxide (H2O2), hydrofluoric acid (HF), and some semivolatile organics including PCBs, DDT, and lindane (see Chapter 7). Particulate deposi- tion onto leaf surfaces brings other particulate sorbed pollutants into contact with plant surfaces, including heavy metals, radionuclides, polynuclear aromatic hydro- carbons (PAHs), and sulphur (often associated with metals), which become bound to leaf cuticles (Veijalainen, 1988). In addition, clogging of stomatal pores by fine particulates can adversely affect plant CO2and water regulation. Rainfall interacts with vegetation surfaces modifying their original composition, leaching elements and ions released by plant organs, and washing out powders and aerosols deposited on the leaf surfaces. These substances may be transferred to the soil where some of them become available to the root systems of plants (e.g., metals). Throughfall and stemflow are the most important vectors of pollutants and nutrients from the atmosphere to the soil.

Pollutants first come into contact with the epidermal layer, the interface between the inside and outside of the plant. Ozone affects the cuticle by vigorously react- ing with components such as hydroxy fatty acids therein, stripping away leaf waxes. However, cuticular transpiration is not affected by O3. Ozone enters leaves via stomata and is received at the apoplastic space where its concentration decreases, indicating some degree of detoxification. Atmospheric photooxidation products such as peroxyacetyl nitrate (PAN) also enter plants via stomata, moving into the substomatal space and dissolving in the extracellular fluid of cells. PANs are trans- ported by the transpiration stream to developing areas of leaves, damaging sul- phydryl groups of proteins and unsaturated double bonds of lipids. About 90% SO2 is absorbed by stomata. Entry of SO2 into leaves is abnormal (sulphur normally enters via the roots). Ammonia is codeposited with SO2.

A summary of routes of uptake of various toxicants by plants is provided in Table 3.1.

3.3

Uptake at the tissue and cellular level

In rare cases, chemicals may exert toxic action without entering the tissues. For example, excess mucus production by fish may occur in reaction to the presence of aluminium at the gill epithelial surface, thereby inhibiting gaseous exchange. However, generally speaking the toxicant needs to cross both aqueous and lipid compartments of the integument. The first barrier encountered is the plasma mem- brane of the epithelial cells lining the integument. This is a typical biomembrane about 80 Å thick consisting of a lipid biolayer and a variety of proteins, some of which may function as transporters. Water and small organic molecules may diffuse across the membrane through small pores (0.2–0.4 nm diameter), although these pores are relatively few in number. The nonpolar regions of the lipids and proteins

Uptake at the tissue and cellular level 79

Table 3.1. Uptake routes for various toxicants in plants

Toxicant Routes of entry of toxicants to plants SO2 90% absorbed by stomata.

NH3/NH4 50% wet/50% dry deposition found in most European countries. Wet deposited N taken up by roots. NH3entering leaf stomata dissolves in leaf to form NH4.

CO Stomatal uptake incorporated into CO2uptake routes (Wellburn, 1994).

F Toxicant occurs as particulate or gaseous HF. Enters old or weathered leaves via stomata, dissolves in water surrounding mesophyll cell, and is transported in transpiration stream to leaf tips and margins causing necrosis.

H2O2 Route of entry is principally gaseous in stomata. Toxicant is received in a localised place called the apoplastic space in aqueous matrix of cell walls.

Semivolatile The pattern of uptake depends on Henry’s law constant (relating to organics volatility) and octanol:water partition coefficient (relating to lipid

solubility). Mono- to tetrachlorobiphenyls are transferred to plants in gaseous form. Higher chlorinated PCBs sorbed to particulates and aerosols are transferred to plants by depositional processes and revolatilisation as gases in hot summer months. Stomatal entry and solubilisation in epicutical waxes are important entry routes of many semivolatile organics and PCBs.

Bromacil, High water solubility and low Henry’s law constants dictate root 2,4-D uptake from soil and transfer to leaves in transpiration stream (herbicides) (Paterson et al., 1994).

Mercury Toxicant transported to plants in free gaseous form (as well as particulate bound) where it is oxidized and removed by wet and dry deposition processes to ionic and particulate species (Hacon et al., 1995).

Trace metals Toxicant principally taken up from soil by roots but also through bark from smelting into wood or through leaves and transported via phloem to xylem. activities

Radionuclides Absence of cuticle in lichens permits favourable uptake, particularly (e.g., 134, 137

Cs, in foliose forms (e.g., Xanthonia) and species with large flat thalli 210_Pb, 210_{Po) (e.g., Peltigera). Lichens lack root system and uptake is principally}

from the atmosphere (ombrotrophic) by deposition and mechanical trapping (e.g., Pyatt and Beaumont, 1989).

consist of the long hydrocarbon chains of the lipids and the nonpolar side chains of the constituent amino acids of the proteins. These comprise the bulk of the mem- brane. Hydrated polar heads of the phospholipids and polar amino acid side chains of the protein constitute the surface of the membrane (Figure 3.2C) and provide

compatible interaction with aqueous compartments on either side. Hydrogen bonding in these aqueous phases provides support for the main structure of the membrane.

3.3.1 Toxicokinetics

DIFFUSION

Toxicants that penetrate the nonpolar portion of the membrane do so by a process of passive diffusion. Nonpolar organic compounds are highly lipid soluble and cross lipid membranes easily. This tendency to move from the aqueous to the lipid phase (hydrophobicity) is conveniently modelled by the octanol : water partition coefficient. Passage of nonpolar chemicals through the lipid portion of the mem- brane is principally limited by the molecular size and lipid solubility of the com- pound. Other limitations include the thickness and surface area of the membrane and the overall diffusive process is defined by Fick’s law:

(3.1)

where D is the diffusion rate, C0/Ciis the concentration gradient, MW is the mol- ecular weight of the diffusing chemical, s is its solubility in the membrane, and A and d are the membrane surface area and thickness, respectively.

In respiratory epithelia such as gills and lungs, where A may be very large and

d very small, the solubility of the chemical in the membrane may become much

less important, and in terrestrial animals exposed to gaseous compounds, s is better represented by blood solubility, which would limit absorption through respiratory epithelia.

COMPARTMENTAL MODELS

As we have seen, toxicant uptake can occur through direct absorption from ambient media such as air or water or through the ingestion of contaminated food. After ingestion, toxic chemicals follow a variety of pathways. They may enter short-term storage (i.e., tissues where they have a limited residence time), enter long-term tissue storage, or undergo rapid elimination or metabolism.

Interest in the uptake and retention of toxic substances stems from concern over their toxicity to the organism and their potential for passing toxicants through dif- ferent trophic levels. The kinetics of toxic chemical transfer through food webs are further mentioned in later chapters (Sections 4.5.1, 4.5.2, and 5.4.1).

Here we describe some of the models that have been developed to describe the kinetics of chemical bioaccumulation by an individual organism. Models tend to be either empirical or mechanistic in nature, although many integrate these two approaches. Typical are the compartmental models developed initially by pharmacologists. D C C s A d i =[ ] ¥ ¥ ¥ 0 1 2 MW

Uptake at the tissue and cellular level 81

3.3.2 Single-compartment model

Bioaccumulation of a toxicant by an organism can be written as a mass balance equation wherein the net rate of accumulation is the difference between the uptake and loss (elimination) rates:

(3.2) where CBis the toxicant concentration in the organism, CMis the concentration in the ambient medium, and k1and k2are rate constants for uptake and loss, respec- tively (Figure 3.4).

Integration of (3.2) gives

(3.3) where CBis the toxicant concentration in the organism at time t.

At low concentrations of toxicant, uptake is a first-order process wherein the rate of accumulation is proportional to the external concentration (Figure 3.5). As the toxicant concentration increases, a steady state is reached wherein the rate of uptake approaches the loss rate and the concentration in the organism reaches a plateau. At this point,

(3.4) and

(3.5) The system is now in a state of zero-order kinetics.

Following termination of toxicant exposure, the rate of uptake, k1CM, falls to zero, and the elimination term becomes

(3.6) If this is integrated, (3.7) C C e k t B= 0 - 2 dC dt k C B B = - 2 dC dt k C k C B M B = 1 - 2 =0 k C1 M =k C2 B C k k C e k t B= M◊

(

)

Figure 3.4 Single-compartment model. CB, CM= toxicant concentrations

in body and external medium, respectively. k1, k2= rate constants for

where C0is the concentration in the organism at the beginning of the elimination process. The logarithmic form of this equation,

(3.8) plots as a straight line with a slope of -k2/2.3 (Figure 3.6).

The conventional way of expressing this information is in the form of biologi- cal half-life (t1/2). This is the time taken for an organism to clear half of the toxi- cant content of its body. In a single-compartment system, it is the time for C0to be reduced by half (i.e., at t1/2, C = C0/2). Thus,

(3.9) and (3.10) t k k 1 2 2 2 2 0 693 =ln = . lnC0 lnC0 kt1 2 2 = - log log . CB= C0- k t 2 2 3

Figure 3.6 Kinetics of toxicant elimination from a single (plasma) compart-

ment. CP= toxicant concentration in plasma; toxicant concentration in plasma

at time zero (i.e., t= 0). k2= elimination constant.

Figure 3.5 Rate of toxicant uptake versus toxicant concentration as described

Uptake at the tissue and cellular level 83

In multicelled organisms, the single-compartment model/system is a special case that is only approximated under certain circumstances, for example, (a) where the toxicant remains unchanged in the circulatory fluid and is not (or only very slowly) taken up by the tissues or (b) where the toxicant freely diffuses through- out the blood and tissues without any rate-limiting diffusional barrier.

3.3.3 Two-compartment model

Often the time-course of toxicant concentration in blood plasma following its rapid introduction (e.g., by injection) is a curve rather than a straight line. Such a curve is the result of toxicant distribution into more than one compartment. The simplest case is the two-compartment system where the toxicant distributes rapidly into the plasma and tissues, but its excretion and metabolic transformation (collectively termed elimination) proceed more slowly. The differential equation for this model is

(3.11) where CPand CTare toxicant concentrations in plasma and tissues, respectively;

k2= [ke+ km]; and ke, km, k12, and k21are, respectively, the first-order rate constants for elimination, metabolic transformation, distribution from plasma to tissues, and distribution from tissues to plasma. A schematic representation of this model is shown in Figure 3.7.

Integration gives

(3.12) where A, B, a, and bare complex constants of k12, k21, and k2; in other words,

(3.13) (3.14) and

(3.15) A semi-logarithmic plot of CPversus time gives a biphasic curve with two linear portions, a steep distribution phase with slope aand a more shallow elimination

a+ =b k12+k21+k2 B=C0◊(k12-b a) -b A=C0◊(a-k12) a-b C Ae t Be t P = + -a -b dC dt k C k C k C P T P P = 21 - 12 - 2

Figure 3.7 Two-compartment model. W = waste (excretion); M = metabolism;

k12= rate constant plasma Æ tissues; k21= rate constant tissues Æ plasma;

phase with slope b (Figure 3.8). In the integrated curve, the initial portion repre- sents both distribution and elimination, whereas the terminal portion represents elimination only. The elimination curve may be extrapolated to t = 0, and this can be separated from the initial portion of the curve to enable characterisation of the distribution phase only. A and B are the ordinate intercepts of the distribution and elimination phases, respectively. Thus, a, b, A, and B may be derived graphically, and from these values k12, k21, and k2may be determined as follows:

(3.16) (3.17) (3.18) Although it might be considered that the characteristics of these elimination curves would be governed by gross molecular properties such as size, shape, and electrical charge, sometimes relatively minor changes in molecular configuration may have significant effects on the elimination kinetics. Figure 3.9 illustrates the effect of altering the position of a chlorine atom on a chlorinated aniline on its elimination from zebra fish, Brachydanio rerio (see Table 5.3). A shift in the chlo- rine from the ortho to the meta position significantly extends the distribution phase, suggesting a larger volume of distribution (see following discussion) relative to the ortho form (indicated by the dashed line in Figure 3.9).

In some cases, several different tissues (each with different rate constants) might be substituted for the single-tissue compartment described earlier. If, for any tissue, the rate constant from blood to tissue (k12) = the rate constant from tissue to blood (k21), that tissue can be functionally combined with blood as a single compartment. Beginning with a single source of toxicant available to the organ- ism, the relative changes in toxicant dose occurring in compartments P (plasma), T (tissues), M (metabolised toxicant), and W (excreted toxicant) are shown in Figure 3.10. k k 2 21 =ab k12 =a+ -b k21(-k2) k A B A B 21= + + b a

Figure 3.8 Kinetics of toxicant elimination from a two-compartment system.

As in Figure 3.6. A, B= ordinate intercepts for distribution and elimination phases; µ, b = slopes of distribution and elimination phases.

Uptake at the tissue and cellular level 85

Changes in the various rate constants have a marked effect on the shape of these curves. In classical pharmacokinetic studies, much can be learned by monitoring the central (blood) compartment in a human or a laboratory animal. Figure 3.11 shows the consequences of changing different parameters in a toxicokinetic study. The ability of a chemical to exchange between the blood compartment and one or more tissue compartments owes much to its physical and chemical characteristics. A large, relatively hydrophilic molecule is much more likely to remain in the blood- stream than small, hydrophobic molecules, which will be rapidly distributed to tissue lipids. This phenomenon is addressed through a concept known as volume of distribution.

Figure 3.9 Effect of change in molecular configuration of chlorinated aniline

on elimination from the zebra fish, Brachydanio rerio (after Kalsch et al., 1991).

Figure 3.10 Relative changes in a single toxicant dose in different body com-

partments during uptake. P = plasma compartment; T = tissue compartment; M + W = sum of metabolism and waste compartments.